JP6163467B2 - Natural antisense and non-coding RNA transcripts as drug targets - Google Patents

Description

  The present invention provides compositions and methods for highly selective targeting of heterologous nucleic acid sequences. Oligonucleotides are siRNAs that bind to their target genes in a sequence-dependent manner and control the expression of unwanted nucleic acid sequences in target cells. Furthermore, siRNA can specifically and selectively kill bacterial cells or human cells if the target is present in its genome.

  Over the last few years, the understanding of complex mammalian transcription has increased significantly and many novel ribonucleic acid (RNA) transcripts have been detected. This is because the total number of conventional (protein-encoding) genes in the human genome (approximately 20,000-25,000) is much lower than expected several years ago, and the number of genes in simpler organisms such as Drosophila melanogaster or Caenorhabditis elegans It was somewhat unexpected (Finishing the euchromatic sequence of the human genome. Nature 431 (7011): 931-945 (2004)).

  Two major transcriptomics efforts have complementarily paved the way for a new and revised view of mammalian transcription. First, the international FANTOM (Functional Annotation of the Transcriptome of Mammals) has been producing and analyzing large amounts of complementary DNA (cDNA) sequencing data from human cells and tissues, although mainly from mice for several years. (Carninci, P. et al. (2005) Science 309 (5740), 1559-1563, Katayama, S. et al. (2005) Science 309 (5740), 1564-1566). Independently, high-density (tiling) microarray experiments provide complementary evidence that transcription occurs extensively throughout the human genome and that there are many transcripts of unknown function that are not annotated. (Cheng, J. et al. (2005), Science 308 (5725), 1149-1154, Kapranov, P. et al. (2005) Genome Res 15 (7), 987-997).

  RNA can be classified into (1) messenger RNA (mRNA) that is translated into protein and (2) non-protein-encoding RNA (ncRNA). Until recently, there were only a few ncRNAs (eg tRNA, rRNA and spliceosomal RNA), all of which were thought to be associated with protein synthesis or function. Furthermore, until several years ago, no systematic effort was made to identify new ncRNA transcripts and elucidate their function.

Overview Sequence-specific siRNAs bind to target nucleic acid molecules and control the expression of gene expression products. Methods are provided in which up-regulation or inhibition of the target nucleic acid is effective in treating abnormal cell proliferation, neurological disease, abnormal cell control, disease, and the like. Drug discovery methods are within the scope of the present invention.

  In a preferred embodiment, the method of upregulating gene expression comprises targeting a nucleic acid molecule to an antisense transcript of a sense strand (wherein the nucleic acid molecule targeting the antisense transcript is complementary to the antisense strand). And) binding the nucleic acid to the antisense transcript, thereby increasing the expression of the sense strand and up-regulating the expression of the gene. Preferably, binding of the nucleic acid molecule to the antisense strand is effective under high, moderate or low stringency conditions. Any disease, abnormality (congenital or non-congenital), tumor, pathogen (eg prion, virus, eg tumor-causing virus), molecular target, eg both intracellular and extracellular molecules (eg receptor, enzyme, cytokine transcription) Can be treated by designing molecules that target the desired sense and / or antisense loci. Sequence specific RNA molecules can be designed such that they target overlapping sequences and also target complementary strands.

  In another preferred embodiment, the nucleic acid molecule is an RNA molecule and comprises at least one of SEQ ID NOs: 1-67.

  In another preferred embodiment, the method of inhibiting gene expression comprises targeting a nucleic acid molecule to an antisense transcript and a sense strand transcript, wherein the nucleic acid molecule that targets the antisense transcript and the antisense strand Nucleic acid molecules that are complementary and that target the sense transcript are complementary to the sense strand), and bind the nucleic acid molecule to the antisense and sense transcripts, thereby inhibiting gene expression. Preferably, the nucleic acid molecule is an RNA molecule, and the nucleic acid molecules targeting the antisense and sense transcripts bind and / or overlap in a convergent orientation, a divergent orientation. doing.

  In another preferred embodiment, the nucleic acid molecule is at least one of SEQ ID NOs: 4-8 and the targeted nucleic acid is a coding or non-coding transcript.

  In a preferred embodiment, the target gene is CD97, TS-α, C / EBP delta, CDC23, PINK1, HIF1α, Gnbp3g, adrenomedullin AM1 receptor, 6320439J10 (3-oxoacid CoA transferase), CtpW85 (cathepsin W), Ddx -39, rTS-α, I530027A02, Kif20a, PINK-AS, aHIF1α, Gnbp3g-AS, AdmR-AS, A230019L24 or CtpW-AS. Preferably, the target nucleic acid comprises the nucleic acid sequence PINK-AS, aHIF1α, Gnbp3g-AS, AdmR-AS, A230019L24 or CtpW-AS. Any coding and non-coding sequences, transcripts, and / or sequences that overlap with either the 5'-3 'sequence and the complementary 3'-5' sequence can be targeted.

  In a preferred embodiment, the non-coding nucleic acid targeted is the nucleic acid sequence CD97, TS-α, C / EBP delta, CDC23, PINK1, HIF1α, Gnbp3g, adrenomedullin AM1 receptor, 630439J10 (3-oxo acid CoA transferase), CtpW85 (cathepsin) W), Ddx-39, rTS-α, I530027A02, and Kif20a.

  In another preferred embodiment, the pharmaceutical composition comprises at least one of SEQ ID NOs: 1-67 and variants thereof.

  In yet another preferred embodiment, the isolated nucleic acid comprises any of SEQ ID NOs: 1-67 and variants thereof.

  In another preferred embodiment, the composition comprises CD97, TS-α, C / EBP delta, CDC23, PINK1, HIF1α, Gnbp3g, adrenomedullin AM1 receptor, 6320439J10 (3-oxoacid CoA transferase), CtpW85 (cathepsin W), Ddx A nucleic acid sequence targeting at least one gene and / or transcript comprising -39, rTS-α, I530027A02, Kif20a, PINK-AS, aHIF1α, Gnbp3g-AS, AdmR-AS, A230019L24 or CtpW-AS. The targeted locus may be an overlapping sequence and / or may target both the sense and antisense strands. In other aspects, coding and non-coding sequences are targeted.

  In a preferred embodiment, the method of treating Parkinson's disease comprises administering at least one of SEQ ID NOs: 1-3 to a patient in need thereof.

  In another preferred embodiment, the pharmaceutical composition comprises at least one of the nucleic acid molecules of SEQ ID NOs: 1-67 and variants thereof in a pharmaceutically acceptable carrier.

  In another preferred embodiment, the nucleic acid molecule comprises a siRNA molecule that targets the overlapping region of the sense / antisense locus. For example, a pharmaceutical composition for treating Alzheimer's disease targets a BACE transcript. Nucleic acid molecules can be designed to target overlapping sequences or sequences or loci of BACE transcripts. These molecules specific for overlap targets in the treatment of Alzheimer's disease include SEQ ID NO: 41-43 and SEQ ID NO: 44-45 and variants thereof.

  In another preferred embodiment, the expression vector comprises any one or more of SEQ ID NOs: 1-67 and variants thereof.

  In another preferred embodiment, the isolated peptide is encoded by any one or more of SEQ ID NOs: 1-67 and variants thereof.

  In another preferred embodiment, the isolated antibody is specific for BACE-1 mRNA, BACE-1-AS RNA, SEQ ID NOs: 1-67 and peptides thereof.

  In another preferred embodiment, the method of treating a neurological disease comprises administering to a patient in need thereof at least one of SEQ ID NOs: 4-8, 40-61 and 64-67 and variants thereof. . In a further aspect of the method, SEQ ID NOs: 4-8, 40-61, 64-67 are administered in combinations that target the overlap region of the sense / antisense locus.

  In another preferred embodiment, various combinations of at least two of SEQ ID NOs: 4-8, 40-61, 64-67 and variants thereof are administered to the patient during the treatment period. Examples of neurological diseases include, but are not limited to, Alzheimer's disease, aphasia, Bell's palsy, Creutzfeldt-Jakob disease, epilepsy, encephalitis, Huntington's disease, neuromuscular disorder, neuro-oncology, neuroimmunology, neuro-otology, pain, fear Illness, sleep disorders, Tourette syndrome, Parkinson's disease and other movement disorders.

  In another preferred embodiment, the method of upregulating gene expression comprises targeting a nucleic acid molecule to an antisense transcript of a sense strand, wherein the nucleic acid molecule targeting the antisense transcript is complementary to the antisense strand. And binding the nucleic acid to the antisense transcript, where sense strand expression is elevated and gene expression is upregulated. Preferably, the nucleic acid molecule is an interfering RNA molecule.

  In a preferred embodiment, the nucleic acid molecule is at least one of SEQ ID NOs: 1-3, 4-32, 40-61, 64-67 and variants thereof. In one aspect, the nucleic acid molecules of SEQ ID NOs: 1-3, 4-32, 40-61, 64-67 and variants thereof comprise at least one modified nucleobase.

  In another preferred embodiment, the method of inhibiting gene expression comprises targeting a nucleic acid molecule to an antisense transcript and a sense strand transcript, wherein the nucleic acid molecule that targets the antisense transcript and the antisense strand Nucleic acid molecules that are complementary and that target the sense transcript are complementary to the sense strand), and binding the nucleic acid to the antisense and sense transcripts, where gene expression is inhibited.

  In preferred embodiments, nucleic acid molecules that target the antisense and sense transcripts bind or overlap in a direction that converges or diverges with the transcript. Preferably, the target nucleic acid is a coding and / or non-coding transcript. Examples of targeted genes include: CD97, TS-α, C / EBP delta, CDC23, PINK1, HIF1α, Gnbp3g, adrenomedullin AM1 receptor, 630439J10 (3-oxo acid CoA transferase), CtpW85 (cathepsin W) , Ddx-39, rTS-α, I530027A02, Kif20a, PINK-AS, aHIF1α, Gnbp3g-AS, AdmR-AS, A230019L24 or CtpW-AS. Examples of target encoding nucleic acids include the nucleic acid sequences PINK-AS, aHIF1α, Gnbp3g-AS, AdmR-AS, A230019L24 or CtpW-AS. Examples of non-coding nucleic acids to be targeted are the nucleic acid sequences CD97, TS-α, C / EBP delta, CDC23, PINK1, HIF1α, Gnbp3g, adrenomedullin AM1 receptor, 63043939 (10-oxo acid CoA transferase), CtpW85 (cathepsin W) , Ddx-39, rTS-α, I530027A02, and Kif20a.

  In another preferred embodiment, treating age-related macular degeneration comprises administering to a patient at least one of SEQ ID NOs: 1-67 and variants thereof.

  In another preferred embodiment, the composition comprises CD97, TS-α, C / EBP delta, CDC23, PINK1, HIF1α, Gnbp3g, adrenomedullin AM1 receptor, 6320439J10 (3-oxoacid CoA transferase), CtpW85 (cathepsin W), Ddx -39, rTS-α, I530027A02, Kif20a, PINK-AS, aHIF1α, Gnbp3g-AS, AdmR-AS, A230019L24 or a nucleic acid sequence targeting the coding and noncoding sequences of CtpW-AS including.

  Other aspects of the invention are described below.

BRIEF DESCRIPTION OF THE DRAWINGS The invention is set forth with particularity in the appended claims. The above and further advantages of the present invention may be better understood by reference to the following description taken in conjunction with the accompanying drawings.

  1A and 1B are schematic diagrams showing the basics of antisense transcription. FIG. 1A shows how antisense transcripts can be generated in a mammalian transcriptome. FIG. 1B shows that there are three possibilities for the overlap of the antisense transcript with the sense transcript.

  FIGS. 2A-2B are schematics illustrating two possibilities for altering sense transcript levels by causing antisense knockdown (eg, by using siRNA). FIG. 2A represents the case where the controls are discordant, while FIG. 2B illustrates the concomitant knockdown of both the sense transcript and the corresponding antisense transcript.

  FIG. 3A is a schematic representation of thymidylate synthase (TS-sense) and rTSα antisense mRNA. The exon boundaries are marked with a crossing bar. The position of the probe used for TaqMan and in situ hybridization, as well as the 3 'overlap region of S-AS mRNA are also shown. FIG. 3B shows the structure of the vector used for transfection and S-AS RNA production. The sense vector generates RNA having a 3 ′ sense sequence, the antisense vector generates RNA having a 3 ′ antisense sequence, and the S-AS vector has a hairpin between the sense RNA sequence and the antisense RNA sequence. An RNA having a continuous sense-antisense sequence with the sequence is generated.

  FIG. 4 is a fluorescence image showing single cell RNA expression of the TS transcript. (A) antisense probe, (b) sense probe and (c) sense and antisense probe bound to fixed and reverse transcribed TS RNA in HeLa cells. The probe was designed to cover the exon boundaries and part of the overlap region in a strand specific manner. (D) The signal from the actin probe indicates that the method was functioning optimally. All probes straddled introns to avoid background signal from contaminating DNA.

  FIG. 5 is a graph showing endogenous single cell mRNA expression of TS sense and antisense molecules. Real-time PCR primers were designed to span between overlapping and non-overlapping regions. To measure the sensitivity of the assay, the expression of trace amounts of TATA box binding protein was also quantified. All samples were normalized to β2-microglobulin and the average of results from 15 individual cells was plotted.

  FIG. 6 shows the cellular localization of TS sense (TS) and its antisense (rTSα), HIF sense (HIF) and its antisense (aHIF) in three cell lines (HeLa, SK-N-MC and HEPG2). It is the shown graph. Cytoplasm and nuclear RNA were normalized to total RNA.

  FIG. 7 is a blot showing RPA of cytoplasmic RNA. Lane 1: HeLa cell cytosolic lysate. Lane 2: cytoplasmic lysate of HeLa cells overexpressing S-AS. Lane 3: Lysate from HeLa cells transfected with S-AS RNA duplex transcribed in vitro. Lane 4: Total RNA from HeLa cells overexpressed S-AS. All were treated with RNAse A + T, separated by denaturing PAGE and probed for overlapping regions of thymidylate synthase mRNA.

  FIG. 8 shows a Northern blot for the Dicer product. Lane (a): Total RNA from HeLa cells, Lane (b): Total RNA from HeLa cells overexpressed S-AS mRNA, Lane (c): From HeLa cells transfected with IVT-overlapping dsRNA , Total lane (d): total RNA from HeLa cells overexpressing the continuous S-AS RNA (m) marker.

  FIG. 9 is a graph showing the expression of IFNβ and OAS2 mRNA. Interferon responses were quantified by qRT-PCR in HeLa cells transfected with IVT-dsRNA TS or overexpressed S-AS and reference HeLa. mRNA levels were normalized to GAPDH.

  FIGS. 10A-10B are graphs showing expression profiles of BACE-1 and BACE-1-AS in various regions of mouse brain and liver. All transcripts were 2-5 times more numerous in various regions of the brain compared to the liver. The cerebral cortex and amygdala had the highest expression of both transcripts in the sampled brain area (FIG. 10A). Northern blot analysis confirmed the expression of BACE-1 and BACE-1-AS in mouse brain tissue (FIG. 10B). BACE-1 and BACE-1-AS transcripts were also expressed in undifferentiated and differentiated human neuroblastoma SH-SY5Y cells. Induction of differentiation of human neuroblastoma SH-SY5Y cells is accompanied by a reduction of approximately 50% in BACE-1-AS transcript expression and approximately 20% in BACE-1 transcript expression (FIG. 10B). It was suggested that the expression of both sense and NAT transcripts was consistent.

  FIGS. 11A and 11B are scanned images of BACE-1 protein Western blots after treatment of SH-SY5Y cells with siRNA. Transfected with BACE-1 protein from control cells (lane-1) and 20 nM siRNA against BACE-1 (lane-2), BACE-1-AS (lane-3) or both transcripts (lane-4) The cells were compared.

  Figures 12A-12d are schematic diagrams showing the genomic organization of the human BACE-1 locus at the human chromosome 11q23.3 locus, the BACE-1 mRNA and BACE-1-AS positions on the genome, and transcription of both It shows a pattern that is strongly conserved in several other species, including mice (Figure 12A). BACE-1 is transcribed from the positive strand of chromosome 11 and BACE-1-AS is transcribed from the negative strand of chromosome 11 to cover exon-6 of BACE-1. FIG. 12B shows the transfer direction and the overlap area between BACE-1 and BACE-1-AS in an enlarged field of view. The sites numbered 1, 2 and 4 are BACE-1 siRNA target sites, and site 3 is a northern blot probe site in the overlap region and capable of detecting both transcripts. Sites 2 and 5 also represent sense and antisense FISH probe sites, respectively. Sites 6, 7 and 8 are all BACE-1-AS siRNA target sites in the non-overlapping part of the AS transcript. Sites 5 and 9 represent the 3 'and 5' RACE primer sites, respectively. Figures 12C and 12D represent the RACE sequencing data for BACE-1-AS from human and mouse brain, respectively (16 clones each). Sequencing data revealed that the antisense transcript contains a cap structure and a poly A tail and undergoes differential splicing in both humans and mice. The yellow highlighted area is the overlapping region to the BACE-1 sense transcript, and the green highlighted area is newly derived from our sequencing data and these have existed in the genomic database so far. There wasn't. Point mismatches to the genome sequence are indicated by an asterisk (*) for changes from A to G and crosses (†) for changes from C to T.

  Figures 13A and 13B are graphs showing silencing of BACE-1 and BACE-1-AS transcripts by synthetic siRNA. FIG. 13A shows that BACE-1 (S-a) siRNA transfection reduced BACE-1 mRNA without significant changes in BACE-1-AS levels. Targeting BACE-1-AS transcripts with three different siRNAs (AS-a, AS-b and AS-c) resulted in significant down-regulation of both BACE-1 and BACE-1-AS transcripts (P <0.0001). SiRNA 20 nM targeting the non-overlapping portion of the transcript was transfected into neuroblastoma cells (SH-SY5Y). All samples were normalized to 18s rRNA and graphed as a percentage of each mRNA relative to the negative control siRNA sample. FIG. 13B shows detection of amyloid Aβ1-42 protein by ELISA. HEK-SW cell supernatants were analyzed for amyloid Aβ1-42 protein after transfection of 20 nM siRNA against BACE-1 (Sa), BACE-1-AS (AS-a) or both. Results were plotted against control siRNA transfected cells. Aβ1-42 protein was significantly downregulated by siRNA targeting BACE-1 or BACE-1-AS (P <0.0001). The combination of both siRNAs (10 nM each) caused a more significant down-regulation (P <0.001).

  14A-14E are graphs showing in vivo injection of unmodified synthetic siRNA into mouse brain. Unmodified synthetic siRNAs were designed to target non-overlapping regions of either BACE-1 (sense) or BACE-1-AS for degradation. Control siRNA has similar properties and has previously been shown to have no effect on human and mouse genes. Three groups of mice were subjected to continuous infusion of siRNA for 2 weeks. Five tissues from each mouse were used for quantitative measurement of RNA by real-time PCR. Fig. 14A: Dorsal medial prefrontal cortex (PFC), Fig. 14B: ventral hippocampus, Fig. 14C: dorsal hippocampus, Fig. 14D: dorsal striatum, and Fig. 14E: cerebellum. SiRNA against either BACE-1 or BACE-1-AS transcript resulted in a simultaneous decrease in both BACE-1 and BACE-1-AS levels compared to the control treatment group (** * = P <0.0001). Both transcripts in the cerebellum (e) were unchanged (P = 0.1518), as expected for tissues not directly connected to the third ventricle of the brain.

  Figures 15A-15F show stress induced relocation of antisense transcripts. FIG. 15A is a scanned image of a photograph showing the distribution of BACE-1 and BACE-1-AS RNA to the nucleus versus the cytoplasm. In RNA-FISH images from neuroblastoma cells SH-SY5Y, the upper panel shows DAPI nuclear staining, BACE-1 mRNA signal covering the entire cell (Alexa Flour 594) and overlay images of both from left to right. Show. The lower panel shows, from left to right, DAPI nuclear staining, mainly a BACE-1-AS signal (Alexa Flour 488) that can be detected inside the nucleus and an overlay image of both. RT-PCR data show that BACE-1-AS transcript is present in the nucleus about 30 times more in the nucleus than the cytoplasm of SH-SY5Y cells, while β-actin and BACE-1 mRNA (sense) are present in both compartments. Clarified that they are almost equal. FIG. 15B shows a scanned image of a photograph showing that the observed nuclear retention pattern of BACE-1-AS is substantially lost when SH-SY5Y cells are exposed to hyperosmotic shock with 30 nM KCl for 5 minutes. It is. When the cells were washed with PBS and returned to normal medium for 1 hour, nuclear retention was restored again. Image shows BACE-1-AS signal translocation from SH-SY5Y nucleus to cytoplasm when exposed to KCl for 5 min, left to right, DAPI nuclear staining, detected from whole cells A possible BACE-1-AS signal and an overlay image of both. FIG. 15C is a photographic scan image showing that synthetic Aβ1-42 peptide was used as a different stressor for monitoring changes in nuclear retention of BACE-1-AS. When SH-SY5Y cells were exposed to 1 μM Aβ1-42 for 2 hours, it was shown that BACE-1-AS left the nucleus. Relocation was recovered when the peptide was washed and the cells were maintained in normal medium for 1 hour. From left to right, the set of panels representing BACE-1-AS rearrangement with similar exposure is DAPI nuclear staining, BACE-1-AS and overlay images of both. In FIG. 15D, 7PA2-CHO cells have been previously shown to overproduce Aβ1-42 dimers and oligomers. Conditioned media of these cells and control parental CHO cells were collected and replaced with normal media of SH-SY5Y cells for 2 hours. Importantly, only the conditioned medium of 7PA2-CHO cells was able to relocate the BACE-1-AS transcript from the nucleus to the cytoplasm. The set of panels shows a similar rearrangement of BACE-1-AS following incubation with 7PA2 conditioned medium. From left to right, DAPI is an overlay image of nuclear staining, BACE-1-AS and both signals. FIG. 15E is a graph showing that human Aβ1-42 is significantly elevated in the brain of APP-tg mice compared to wild-type, as revealed from the HTRF assay. FIG. 15F shows that an increase in the level of Aβ1-42, as in the in vitro experiment, was a 50% increase in BACE-1-AS (P <0.0001), followed by a consistent increase in BACE-1. It is a graph which shows having produced. The increased level of BACE-1-AS was above BACE-1 (P <0.001).

  FIGS. 16A-16E are graphs showing the involvement of cyclophilin-60 in the localization / regulation of BACE-1-AS and the stability of BACE-1 and BACE-1-AS transcripts. Effective knockdown of Cyp-60 in HEK-293T cells causes significant down-regulation (P <0.001) of BACE-1 and BACE-1-AS (FIG. 16A). FIG. 16B: After treatment of HEK293T cells with Cyp-60 siRNA, BACE-1-AS downregulation occurs at an early time point and begins 6 hours after transfection. FIG. 16C: Cyp-60 siRNA treatment also significantly reduced BACE-1-AS nuclear retention (P <0.0001). FIG. 16D: Stability of BACE-1 and BACE-1-AS transcripts over time was measured by real-time PCR in HEK-293T cells after stopping new RNA synthesis with α-amanitin. BACE-1-AS had a shorter half-life than BACE-1 and β-actin. The product of RNA polymerase III, 18s rRNA, was unchanged and served as an endogenous control. FIG. 16E: Incubation of SH-SY5Y cells with conditioned medium of 7PA2 cells, causing translocation of BACE-1-AS to the cytoplasm, significantly increased BACE-1 stability (P <0.001).

  FIG. 17 is a schematic diagram of BACE-1-AS mediating the regulation of BACE-1 and its proposed mechanism of involvement in the pathophysiology of Alzheimer's disease. BACE-1-AS RNA is primarily localized to the nucleus during unstressed states, through interactions with cyclophilin-60 (Cyp-60) and / or some other unknown protein. Different types of cell stressors can potentially initiate a series of events that lead to the transition of BACE-1-AS, which in turn leads to increased stability of BACE-1 mRNA. Increased levels of BACE-1 promote the production of Aβ1-42, leading to Aβ aggregation and Aβ plaque formation. These late events have been shown to be powerful cell stressors and act as feedforward mechanisms leading to the release of more BACE-1-AS from the nucleus.

DETAILED DESCRIPTION Small interfering RNA (siRNA) knocks down an antisense transcript and controls the expression of its sense partner. This control may be inconsistent (sense transcripts increase with antisense knockdown) or concordant (sense transcripts decrease with antisense knockdown). A novel pharmacological strategy based on knockdown of antisense RNA transcripts by siRNA (or another RNA targeting theory) is provided. In the case of discordant control, knockdown of the antisense transcript is thought to increase expression of the traditional (sense) gene, thereby mimicking the agonist / activator action. In the case of consistent control, simultaneous knockdown of the antisense and sense transcripts synergistically reduces conventional (sense) gene expression.
Definition

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein.

  As used herein, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.

  As used herein, the term “specific oligonucleotide” refers to (i) a sequence that can form a stable complex with a portion of the targeted gene, or (ii) the targeted gene. Refers to an oligonucleotide having a sequence capable of forming a stable duplex with a portion of the mRNA transcript.

  As used herein, the terms “oligonucleotide”, “siRNA”, “siRNA oligonucleotide” and “siRNAs” are used interchangeably herein and refer to deoxyribonucleosides, ribonucleosides, substituted forms thereof and alpha -Includes natural and / or modified monomers or linked linear or circular oligomers, including anomeric forms, peptide nucleic acids (PNA), locked nucleic acids (LNA), phosphorothioates, methylphosphonates, and the like. Oligonucleotides bind specifically to target polynucleotides by a regular pattern of monomer-to-monomer interactions, such as Watson-Crick base pairing, Hoogsteen or reverse Hoogsteen base pairing, etc. can do.

  Oligonucleotides may be “chimeric”, ie, contain different regions. In the context of this invention, a “chimeric” compound is an oligo that contains two or more chemical regions, such as one or more DNA regions, one or more RNA regions, one or more PNA regions, etc. It is a nucleotide. Each chemical region is composed of at least one monomer unit, ie a nucleotide in the case of oligonucleotide compounds. These oligonucleotides typically include at least one region in which the oligonucleotide is modified to exhibit one or more desired properties. Desired properties of the oligonucleotide include, but are not limited to, for example, increased resistance to nuclease degradation, increased cellular uptake, and / or increased binding affinity for the target nucleic acid. Different regions of the oligonucleotide may therefore have different properties. The chimeric oligonucleotide of the present invention can be formed as a mixed structure of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and / or oligonucleotide analogs as described above.

  Oligonucleotides can be linked "in register", i.e. if the monomers are linked together, they can be linked as in natural DNA, or can be linked via a spacer. An area can be included. The spacer is intended to form a covalent “bridge” between the regions, and in preferred cases has a length that does not exceed about 100 carbon atoms. Spacers may have different functionalities, such as having positive or negative charges, unique nucleic acid binding properties (intercalators, groove binders, toxins, fluorophores, etc.), lipophilic and It involves inducing a unique secondary structure, such as an alanine-containing peptide that induces an alpha helix.

  As used herein, the term “monomer” is typically linked by a phosphodiester bond or analog thereof and has a size in the range of several monomer units, such as from about 3-4 monomer units to about several hundred monomer units. The monomer which forms the oligonucleotide is shown. Analogs of phosphodiester linkages include: phosphorothioate, phosphorodithioate, methylphosphonate, phosphoroselenoate, phosphoramidate, etc., and are described more fully below.

In the present context, the term “nucleobase” encompasses naturally occurring and non-naturally occurring nucleobases. It will be apparent to those skilled in the art that various nucleobases that were previously considered "non-naturally occurring" were subsequently found in nature. Thus, “nucleobase” includes not only its known purine and pyrimidine heterocycles, but also heterocyclic analogs and tautomers thereof. Illustrative examples of nucleobases include adenine, guanine, thymine, cytosine, uracil, purine, xanthine, diaminopurine, 8-oxo-N ⁶ -methyladenine, 7-deazaxanthine, 7-deazaguanine, N ⁴ , N ⁴ - ^{Etanoshitoshin,} N 6, ^{N 6} - ethano-2,6-diaminopurine, ^{5-methylcytosine, 5- (C 3 ~C 6)} - alkynyl cytosine, 5-fluorouracil, 5-bromouracil, pseudoephedrine isocytosine 2-hydroxy-5-methyl-4-triazolopyridine, isocytosine, isoguanine, inosine and “non-naturally occurring” nucleobases described in Benner et al., US Pat. No. 5,432,272. The term “nucleobase” is intended to encompass each and all of these examples, as well as analogs and tautomers thereof. Particularly interesting nucleobases are adenine, guanine, thymine, cytosine and uracil which are considered to exist naturally for therapeutic and diagnostic applications in humans.

  As used herein, “nucleoside” includes natural nucleosides, which are in the 2′-deoxy and 2′-hydroxyl forms, eg, Kornberg and Baker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992). Includes those described.

  With respect to nucleosides, an “analog” is a synthetic nucleoside having a modified base moiety and / or a modified sugar moiety, such as Scheit, Nucleotide Analogs, John Wiley, New York, 1980, Freier & Altmann, Nucl. Acid. Res., 1997, 25 (22), 4429-4443, Toulme, JJ, Nature Biotechnology 19: 17-18 (2001), Manoharan M., Biochemica et Biophysica Acta 1489: 117-139 (1999), Freier S., M., Nucleic Acid Research, 25: 4429-4443 (1997), Uhlman, E., Drug Discovery & Development, 3: 203-213 (2000), Herdewin P., Antisense & Nucleic Acid Drug Dev., 10: 297-310 (2000) 2′-O, 3′-C bond [3.2.0] bicycloarabinonucleosides (eg NK Christensen., Et al, J. Am. Chem. Soc., 120 : 5458-5463 (1998)). Such analogs include synthetic nucleosides designed to enhance binding properties such as duplex or triplex stability, specificity, and the like.

  With respect to duplex or triplex formation, the term “stability” generally indicates how strongly an antisense oligonucleotide binds to its intended target sequence, and more specifically, “stability” Shows the free energy of formation of double or triple chains under physiological conditions. Melting temperature in a standard set of conditions (eg, those described below) is a convenient indicator of duplex and / or triplex stability. Preferably, the oligonucleotide of the present invention is measured at a strand concentration of 1.5 μM of the oligonucleotide and the target nucleic acid in 100 mM NaCl, 0.1 mM EDTA and 10 mM phosphate buffer aqueous solution, pH 7.0. And having a melting temperature of at least 45 ° C. Thus, when used under physiological conditions, duplex or triplex formation is substantially advantageous over conditions where the antigen and its target dissociate. It is understood that a stable duplex or triplex may include, in some embodiments, mismatches between base pairs and / or mismatches between base triplets in the case of triplex. Preferably, the modified oligonucleotides of the invention, such as those comprising LNA units, form duplexes and / or triplexes that are perfectly matched with the target nucleic acid.

  As used herein, the term “downstream” means in the direction from the 5 ′ to the 3 ′ end when used in reference to the direction along the nucleotide sequence. Similarly, the term “upstream” means in the direction from 3 ′ to 5 ′ end.

  As used herein, the term “gene” means a gene and all currently known variants thereof and any further variants that may be revealed.

  As used herein, a “variant” of a polypeptide refers to an amino acid sequence in which one or more amino acid residues are altered. A variant may have “conservative” changes in which the substituted amino acid has similar structural or chemical properties (eg, replacement of leucine with isoleucine). More rarely, a variant may have “nonconservative” changes (eg, replacement of glycine with tryptophan). Similar minor variations may also include amino acid deletions or insertions, or both. Guidance for determining which amino acid residues can be substituted, inserted or deleted without loss of biological activity are well known in the art, such as computer programs such as LASERGENE software (DNASTAR ) To find out.

  The term “variant”, when used in the context of a polynucleotide sequence, may include a polynucleotide sequence that relates to a wild-type gene. This definition may also include, for example, “allelic”, “splice”, “species” or “polymorphic” variants. Splice variants may have significant identity with a reference molecule, but generally have a greater or lesser number of polynucleotides due to alternative splicing of exons during mRNA processing. Species variants are polynucleotide sequences that vary between different species. Particularly useful in the present invention are wild type target gene product variants. A variant may result from at least one mutation in the nucleic acid sequence and may result in altered mRNA or a polypeptide that may or may not be altered in structure or function. Any given natural or recombinant gene may not have an allelic form, or may have one or multiple allelic forms. Common mutational changes that give rise to variants are generally due to natural deletions, additions or substitutions of nucleotides. Each of these types of changes can occur alone or in combination with other changes in one or more places in a given sequence.

  The resulting polypeptides generally have significant amino acid identity with each other. A polymorphic variant is a change in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants can also include “single nucleotide polymorphisms” (SNPs) or single nucleotide variations that differ by one nucleotide in the polynucleotide sequence. The presence of SNPs can indicate, for example, a tendency of a population to a disease state, ie, susceptibility or resistance.

  As used herein, the term “mRNA” means one or more mRNA transcripts currently known of the targeted gene, and any additional transcripts that may be revealed.

  By “preferred RNA” molecule is meant any foreign RNA molecule that is therapeutic, diagnostic, or otherwise useful. Such molecules include antisense RNA molecules, decoy RNA molecules, enzymatic RNA, therapeutic editing RNA and agonist and antagonist RNA.

  “Antisense RNA” refers to a non-enzymatic RNA molecule that binds to another RNA (target RNA) by RNA-RNA interaction to alter the activity of the target RNA (Eguchi et al., 1991 Annu. Rev. Biochem. 60, 631-652).

  RNA interference “RNAi” is mediated by double-stranded RNA (dsRNA) molecules that have sequence-specific homology to “target” nucleic acid sequences (Caplen, NJ, et al., Proc. Natl. Acad. Sci. USA). 98: 9742-9747 (2001)). Biochemical studies on cell-free lysates of Drosophila have shown that in some aspects of the invention, RNA-dependent gene silencing mediators are “small interfering” RNA duplexes (siRNA) with 21-25 nucleotides. It is shown that. Therefore, siRNA molecules are preferably used in the method of the present invention. siRNA is derived from the processing of dsRNA by an RNase enzyme known as Dicer (Bernstein, E., et al., Nature 409: 363-366 (2001)). The siRNA duplex product is recruited into a multiprotein siRNA complex called RISC (RNA-induced silencing complex). Without wishing to be bound by any particular theory, it is believed that RISC is then directed to a target nucleic acid (preferably mRNA), where siRNA duplexes interact in a sequence-specific manner, Catalytically mediates cleavage (Bernstein, E., et al., Nature 409: 363-366 (2001); Boutla, A., et al., Curr. Biol. 11: 1776-1780 (2001)). Small interfering RNAs that can be used in accordance with the present invention can be synthesized and used according to procedures that are well known in the art and will be familiar to those skilled in the art. The small interfering RNA used in the methods of the present invention preferably comprises from about 0 to about 50 nucleotides (nt). In examples of non-limiting embodiments, the siRNA can comprise about 5 to about 40 nt, about 5 to about 30 nt, about 10 to about 30 nt, about 15 to about 25 nt, or about 20 to 25 nucleotides.

  Selection of appropriate RNAi is facilitated by using computer programs that automatically align nucleic acid sequences and indicate regions of identity or homology. Such a program is used, for example, by searching a database such as GenBank or comparing nucleic acid sequences obtained by sequencing PCR products. Comparison of nucleic acid sequences from different species allows for the selection of nucleic acid sequences that exhibit an appropriate degree of identity between species. For genes that are not sequenced, Southern blots are performed to allow determination of the degree of identity between the gene in the target species and the gene of the other species. As is well known in the art, it is possible to obtain an approximate measure of identity by performing Southern blots with varying degrees of stringency. These procedures allow the selection of RNAi that exhibits high complementarity to the target nucleic acid sequence in the controlled subject and low complementarity to the corresponding nucleic acid sequence in other species. One skilled in the art understands that there is considerable freedom in selecting the appropriate region of a gene for use in the present invention.

  “Enzymatic RNA” means an RNA molecule having enzymatic activity (Cech, 1988 J. American. Med. Assoc. 260, 3030-3035). Enzymatic nucleic acids (ribozymes) act by first binding to a target RNA. Such binding occurs through the target binding portion of an enzymatic nucleic acid that is held in close proximity to the enzyme portion that acts to cleave the target RNA of the molecule. Thus, enzymatic nucleic acids first recognize and then bind to the target RNA through base pairing, and once bound to the correct site, act as an enzyme and cleave the target RNA.

  “Decoy RNA” means an RNA molecule that mimics the natural binding domain for a ligand. The decoy RNA thus competes with the natural binding target for binding of a specific ligand. For example, overexpression of HIV transactivation reaction (TAR) RNA acts as a “decoy” and efficiently binds to the HIV tat protein, thereby binding to the TAR sequence encoded by the HIV RNA. (Sullenger et al., 1990, Cell, 63, 601-608). This is a specific example. Those skilled in the art will recognize that this is only one example, and that other embodiments can be readily generated using techniques generally known in the art.

  The term “complementary” means that one sequence binds to the other sequence, the 3 ′ end of each sequence binds to the 5 ′ end of the other sequence, and each sequence A, T (U), G If C and C can be joined in anti-parallel orientation, aligning with the other sequence T (U), A, C and G, respectively, it means that the two sequences are complementary. Usually, the complementary sequence of an oligonucleotide has at least 80% or 90%, preferably 95%, most preferably 100% complementarity to a defined sequence. Preferably, alleles or variants thereof can be identified. The BLAST program can also be used to assess such sequence identity.

  The term “complementary sequence”, when referring to a polynucleotide sequence, relates to the base sequence in another nucleic acid molecule according to the base pairing rules. More specifically, this or similar terms refer to single strands that are sequenced or amplified between nucleotides or nucleic acids, eg, between two strands of a double stranded DNA molecule, or with oligonucleotide primers. Refers to hybridization or base pairing with a primer binding site on a nucleic acid. Complementary nucleotides are generally A and T (or A and U), or C and G. Two single-stranded RNA or DNA molecules have at least about 95% of the other strand of nucleotides, with one strand of nucleotides optimally aligned, compared, and with appropriate nucleotide insertions or deletions, Usually said to be substantially complementary when paired with at least 98%, and more preferably from about 99% to about 100%. Complementary polynucleotide sequences can be identified by a variety of approaches including the use of well-known computer algorithms and software, such as the BLAST program.

  With respect to duplex or triplex formation, the term “stability” generally indicates how strongly an antisense oligonucleotide binds to its intended target sequence, and more specifically, “stability” Shows the free energy of formation of double or triple chains under physiological conditions. Melting temperature in a standard set of conditions (eg, those described below) is a convenient indicator of duplex and / or triplex stability. Preferably, the oligonucleotide of the present invention is measured at a strand concentration of 1.5 μM of the oligonucleotide and the target nucleic acid in 100 mM NaCl, 0.1 mM EDTA and 10 mM phosphate buffer aqueous solution, pH 7.0. And having a melting temperature of at least 45 ° C. Thus, when used under physiological conditions, duplex or triplex formation is substantially advantageous over conditions where the antigen and its target dissociate. It is understood that a stable duplex or triplex may include, in some embodiments, mismatches between base pairs and / or mismatches between base triplets in the case of triplex. Preferably, the modified oligonucleotides of the invention, such as those comprising LNA units, form duplexes and / or triplexes that are perfectly matched with the target nucleic acid.

  As used herein, the term “thermal melting point (Tm)” is defined as 50% of the oligonucleotide complementary to the target sequence hybridizes to the target sequence in equilibrium under a defined ionic strength, pH and nucleic acid concentration. Refers to the temperature of soybean. Since the target sequence is generally present in excess, at Tm, 50% of the oligonucleotides are occupied at equilibrium. Typically, stringent conditions are such that for short oligonucleotides (eg, 10-50 nucleotides), a salt concentration of pH 7.0-8.3 and a Na ion concentration of at least about 0.01-1.0 M (or alternatively , Other salts) at a temperature of at least about 30 ° C. Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide.

  The term “stringent conditions” refers to hybridization such that the oligonucleotide hybridizes to its target subsequence, but with little or no hybridization to other sequences. It refers to the conditions for soybeans. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5 ° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.

  The term “target nucleic acid” refers to a nucleic acid (often derived from a biological sample) that is designed to specifically hybridize to an oligonucleotide. Either the presence or absence of the target nucleic acid or the amount of target nucleic acid to be quantified is detected. The target nucleic acid has a sequence that is complementary to the nucleic acid sequence of the corresponding oligonucleotide directed to the target. The term target nucleic acid may refer to a specific subsequence of a larger nucleic acid to which an oligonucleotide is directed, or the entire sequence (eg, gene or mRNA) for which detection of its expression level is desired. The difference in usage is clear from the context.

  As used herein, “pharmaceutically acceptable” ingredients / carriers, etc. are suitable for use in humans and / or animals and have unreasonable adverse side effects commensurate with reasonable benefits / risk rates (eg, No toxicity, irritation, and allergic response).

  As used herein, the term “safe and effective amount”, when used in the method of the present invention, does not have any undue adverse side effects (eg, toxicity, irritation, and allergic response) commensurate with a reasonable benefit / risk. In addition, it refers to the amount of an ingredient sufficient to produce the desired therapeutic response. “Therapeutically effective amount” means an amount of a compound of the invention effective to produce the desired therapeutic response. For example, an amount effective to delay the growth or onset of cancer, sarcoma or lymphoma, or to reduce cancer or prevent metastasis. The specific safe or effective amount is the specific symptom being treated, the physical condition of the patient, the type of mammal or animal being treated, the duration of the treatment, the nature of the combination therapy (if any) And the specific dosage form used and factors such as the structure of the compound or derivative thereof.

  As used herein, “pharmaceutical salt” includes, but is not limited to, basic residues such as mineral or organic acid salts such as amines, acidic residues such as alkali or organic salts such as carboxylic acids. . Preferably, the salt is made using an organic or inorganic acid. These preferred acid salts are chloride, bromide, sulfate, nitrate, phosphate, sulfonate, formate, tartrate, maleate, malate, citrate, benzoate, salicylate, Such as ascorbate. The most preferred salt is the hydrochloride salt.

  As used herein, “cancer” refers to any type of cancer or tumor or malignancy found in mammals, including, without limitation, leukemia, lymphoma, melanoma, carcinoma and sarcoma. Examples of cancer include brain cancer, breast cancer, pancreatic cancer, cervical cancer, colon cancer, head and neck cancer, kidney cancer, lung cancer, non-small cell lung cancer, melanoma, mesothelioma, Ovarian cancer, sarcoma, stomach cancer, uterine cancer and medulloblastoma.

  Further cancers that can be treated with siRNA according to the present invention include, for example, Hodgkin disease, non-Hodgkin lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary platelets , Primary macroglobulinemia, small cell lung tumor, primary brain tumor, gastric cancer, colon cancer, malignant pancreatic insulinoma, malignant carcinoid, bladder cancer, precancerous skin lesions, testicular cancer, lymphoma, thyroid Includes cancer, neuroblastoma, esophageal cancer, genitourinary cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, adrenocortical cancer and prostate cancer. Cancer can also be caused by pathogens such as cervical cancer virus and human papillomavirus. Accordingly, in one embodiment, treatment of the disease comprises treatment of an abnormality induced by a pathogen.

  A “heterologous” component refers to a component that has been introduced or generated in an entity that is different from the entity in which it is naturally located. For example, a polynucleotide derived from one organism and introduced into a different organism by genetic engineering techniques is a heterologous polynucleotide, which, when expressed, can encode a heterologous polypeptide. Similarly, a promoter or enhancer that is removed from its native coding sequence and operably linked to a different coding sequence is a heterologous promoter or enhancer.

  As used herein, “promoter” refers to a polynucleotide sequence that controls transcription of a gene or coding sequence to which it is operably linked. Numerous promoters from a variety of different sources are well known in the art, including constitutive promoters, inducible promoters and repressible promoters, as cloned polynucleotide sequences or portions thereof (E.g., from depositaries such as ATCC, as well as other commercial or personal sources).

  As used herein, “enhancer” refers to a polynucleotide sequence that enhances transcription of a gene or coding sequence to which it is operably linked. Numerous enhancers from a variety of different sources are well known in the art and are available as cloned polynucleotide sequences or as part thereof (eg, depository agencies such as ATCC, as well as others) From commercial or personal sources). A plurality of polynucleotides comprising a promoter sequence (such as the widely used CMV promoter) also comprise an enhancer sequence.

  “Operably linked” refers to the components so described being aligned in a relationship that allows them to function in their intended manner. A promoter is operably linked to a coding sequence if it controls the transcription of the coding sequence. Although an operably linked promoter is generally located upstream of the coding sequence, the promoter is not necessarily adjacent to the coding sequence. An enhancer is operably linked to a coding sequence if it enhances transcription of the coding sequence. The operably linked enhancer can be located upstream of the coding sequence, in the coding sequence or downstream of the coding sequence. A polyadenylation sequence is operably linked to a coding sequence if it is at the downstream end of the coding sequence and transcription is allowed to proceed through the coding sequence to the polyadenylation sequence.

  A “replicon” refers to a polynucleotide comprising an origin of replication that allows the polynucleotide to replicate in a suitable host cell. Examples include target cell replicons (eg, nuclear and mitochondrial chromosomes) into which heterologous nucleic acids can be incorporated, and extrachromosomal replicons (eg, replicating plasmids and episomes).

  “Gene delivery”, “gene transfer”, and the like, as used herein, are exogenous polynucleotides (sometimes referred to as “transgene products”) to a host cell, regardless of the method used for the transfer. It is a term that refers to the introduction of. Such methods include various well-known techniques such as vector-mediated gene transfer (eg, by viral infection / transfection, or by various other protein- or lipid-based gene delivery complexes), and “ Techniques that facilitate delivery of “naked” polynucleotides (eg, electroporation, “gene gun” delivery, and various other techniques used for introduction of polynucleotides). The introduced polynucleotide may be stably or temporarily maintained in the host cell. Stable maintenance is typically that the introduced polynucleotide contains an origin of replication compatible with the host cell, or is integrated into the host cell's replicon, eg, an extrachromosomal replicon (eg, a plasmid) or a nuclear or mitochondrial chromosome. It needs to be done. Multiple vectors are known in the art and are known to be capable of mediating the transfer of gene products to mammalian cells as described herein.

  “In vivo” gene delivery, gene transfer, gene therapy, and the like, as used herein, introduces a vector comprising an exogenous polynucleotide directly into an organism, eg, a human or non-human mammal, thereby exogenous. It is a term that refers to the introduction of a sex polynucleotide into cells of such organisms in vivo.

  A cell is “transduced” by a nucleic acid when it is transferred from the extracellular environment into the cell. Any method of introducing a nucleic acid into a cell can be used, and this term does not imply any specific method of delivering the nucleic acid into the cell unless otherwise specified. A cell is “transformed” by a nucleic acid when the nucleic acid is transduced into the cell and stably replicates. Vectors include nucleic acids (usually RNA or DNA) that are expressed by cells. Vectors optionally include substances that help achieve the entry of nucleic acids into cells, such as virus particles, liposomes, protein coatings, and the like. A “cell transduction vector” is a vector that encodes a nucleic acid that is capable of stable replication and expression once the nucleic acid has been transduced into a cell.

  As used herein, a “target cell” or “recipient cell” or an individual that is or is desired to be an exogenous nucleic acid molecule, polynucleotide and / or protein recipient. Refers to a cell or cell. The term is also intended to include single cell progeny.

  A “vector” (sometimes referred to as a gene delivery or gene transfer “vehicle”) refers to a macromolecule or complex of molecules comprising a polynucleotide that is delivered in vitro or in vivo to a host cell. The polynucleotide to be delivered may comprise a coding sequence that is of interest in gene therapy. Vectors, for example, mediate delivery of viral vectors (eg, adenovirus (“Ad”), adeno-associated virus (AAV) and retrovirus), liposomes and other lipid-containing complexes, and polynucleotides to host cells. Including other macromolecular complexes capable of Vectors can also include other components or functions that further regulate gene delivery or gene expression or otherwise provide beneficial properties to the targeted cells. As described and exemplified in more detail below, such other components include, for example, components that affect binding or targeting to cells (including components that mediate cell type or tissue specific binding), vector nucleic acids by cells A component that affects the uptake of the polynucleotide, a component that affects the localization of the polynucleotide in the cell after the uptake (eg, an agent that mediates nuclear localization), and a component that affects the expression of the polynucleotide. Such components may also include a marker, eg, a detectable and / or selectable marker that can be used to detect and select cells that have taken up and are expressing nucleic acid delivered by the vector. Such components may be provided as natural features of the vector (eg, utilization of some viral vectors with components or functions that mediate binding and uptake), or the vector may be modified to provide such functions. It may be. Other vectors include those described by Chen et al; BioTechniques, 34: 167-171 (2003). A wide variety of such vectors are known in the art and are generally available.

  “Recombinant viral vector” refers to a viral vector comprising one or more heterologous gene products or sequences. Because many viral vectors exhibit size constraints associated with packaging, heterologous gene products or sequences are typically introduced by replacing one or more portions of the viral genome. Such viruses can be replication-deficient and require the provision of a deleted function in trans during viral replication and encapsidation (eg, gene products required for replication and / or encapsidation). Having helper virus or packaging cell line). Modified viral vectors have also been described in which the delivered polynucleotide is carried outside the viral particle (see, eg, Curiel, D T, et al. PNAS 88: 8850-8854, 1991).

  As used herein, viral “packaging” refers to a series of intracellular events that result in the synthesis and assembly of a viral vector. Packaging typically consists of a “proviral genome” or a recombinant provector typically referred to as a “vector plasmid” (typically flanked by appropriate viral “packaging sequences”). , Which is a recombinant polynucleotide that can be packaged in a manner similar to the viral genome, followed by encapsidation or other nucleic acid coating. Thus, when an appropriate vector plasmid is introduced into a packaging cell line under appropriate circumstances, it can be replicated and assembled into a viral particle. Viral “rep” and “cap” gene products found in many viral genomes are gene products that encode replication and encapsidation proteins, respectively. A “replication-deficient” or “replication-incompetent” viral vector is missing or altered one or more functions required for replication and / or packaging, and is a viral vector Refers to a viral vector that is unable to initiate viral replication after uptake by a host cell. In order to generate stocks of such replication-defective viral vectors, viral or proviral nucleic acids can be supplied in trans to a “packaging cell line” that has been modified to contain a gene product that encodes a defective function. Can be introduced. For example, such packaging gene products can be stably integrated into a replicon of a packaging cell line, or they can be transferred by a helper virus carrying a “packaging plasmid” or a gene product encoding a missing function. It can be introduced by

  A “detectable marker gene” is a gene that allows a cell having that gene to be specifically detected (eg, distinguished from cells that do not have the marker gene). A wide variety of such marker gene products are known in the art. Preferred examples include detectable marker gene products that encode proteins that appear on the cell surface, thereby facilitating simple and rapid detection and / or cell sorting. As an example, the lacZ gene encoding β-galactosidase can be used as a detectable marker, and cells transduced with a vector having the lacZ gene can be detected by staining.

  A “selectable marker gene” is a gene that allows cells having that gene to be specifically selected or excluded in the presence of a corresponding selection agent. As an example, an antibiotic resistance gene can be used as a positive selectable marker gene that allows host cells to be positively selected in the presence of the corresponding antibiotic. Selectable markers can be positive, negative or bifunctional. A positive selectable marker allows selection of cells with the marker, while a negative selectable marker allows selective exclusion of cells with the marker. A variety of such marker gene products have been described, including bifunctional (ie positive / negative) markers (eg, WO 92/08796 published May 29, 1992, and December 8, 1994). See WO 94/28143 published on the day). Such marker gene products can provide additional control means that may be advantageous in the context of gene therapy.

  “Diagnostic” or “diagnosed” means identifying the presence or nature of a pathological condition. Diagnostic methods differ in their sensitivity and specificity. The “sensitivity” of a diagnostic assay is the percentage of affected individuals determined to be positive (the percentage of “true positives”). Affected individuals not detected by the assay are “false negatives”. Individuals who are not affected and are determined to be negative in the assay are referred to as “true negatives”. The “specificity” of a diagnostic assay is the 1-false positive rate, where the “false positive” rate is defined as the percentage of individuals who do not have a disease determined to be positive. Individual diagnostic methods may not provide a definitive diagnosis of the condition, but it is sufficient if the method provides a positive indicator to aid diagnosis.

  The term “patient” or “individual” is used interchangeably herein and refers to a mammalian subject to be treated, preferably a human patient. In some cases, the methods of the invention are used in the development of animal models for laboratory animals, veterinary applications, and diseases, including but not limited to rodents including mice, mice and hamsters. , And primates.

  “Treatment” is an intervention performed with the intention of preventing the onset of a disease or altering the pathology or symptoms of a disease. “Treatment” thus refers to both therapeutic treatment and prophylactic or preventative measures. “Treatment” can also be identified as paraactive care. Those in need of treatment include those already with the disease as well as those in which the disease is to be prevented. In the treatment of a tumor (eg, cancer), the therapeutic agent may directly reduce the pathology caused by the tumor cells, or the tumor cells may be made more sensitive to treatment with other therapeutic agents, eg, radiation and / or chemotherapy. Also good.

  “Treatment of a neoplastic disease, neoplastic cell, cancer” can result in one or more of the following effects: oligonucleotides, vectors and / or peptides described herein and in the examples below (1) some inhibition of tumor growth, including (i) slowing down and (ii) complete growth arrest, (2) reducing the number of tumor cells, (3) maintaining tumor size, ( 4) Reduction of tumor size (5) (i) reduction, (ii) deceleration or (iii) inhibition of tumor cell invasion to peripheral organs, including (6) (i) reduction, (ii) Metastasis, including slowing or (iii) complete prevention, (7) (i) maintaining tumor size, (ii) reducing tumor size, (iii) slowing tumor growth, (iv) reducing invasion, slowing And preventing or (v) reducing, slowing and reducing metastases Enhancement of anti-tumor immune response that can lead to stop, and associated with / or (8) diseases, 1 or relieving to some extent two or more symptoms.

  Treatment of an individual afflicted with an infectious organism refers to the reduction and elimination of the disease organism in the individual. For example, viral particle reduction as measured by plaque forming units or other automated diagnostic methods such as ELISA.

  “Neurological (neuronal, neurological) defect, disorder or disease” as used herein refers to any neurological disorder or disease including, but not limited to: neurodegenerative diseases (eg, Parkinson's disease, Alzheimer's disease), or autoimmune diseases of the central nervous system (eg, multiple sclerosis), memory loss, long and short-term memory impairment, learning disorders, autism, depression, benign amnesia, childhood Learning disorders, closed head trauma, and attention deficit disorders, brain autoimmune diseases, neuronal responses to viral infections, brain damage, depression, mental disorders such as bipolarism, schizophrenia, narcolepsy / Sleep disorders (including circadian rhythm disorders, insomnia and narcolepsy), nerve cuts or damage, cerebrospinal nerve (CNS) cuts and brain damage Or any damage to neurons, neurological deficits related to AIDS, tics (eg Gilles de la Tourette syndrome), Huntington's chorea, schizophrenia, traumatic brain injury, tinnitus, neuralgia, Inappropriate neuronal activity, ataxia, muscular stiffness (spasticity) and temporomandibular disorders, specifically leading to neurodysthesias in diseases such as trigeminal neuralgia, neuropathic pain, diabetes, MS and motor neuron disease Neurotoxicity due to Reward Deficiency Syndrome (RDS) behavior, alcohol or drug abuse (eg, ecstasy, methamphetamine, etc.) in the subject. Other diseases include, but are not limited to: aphasia, Bell's palsy, Creutzfeldt-Jakob disease, epilepsy, encephalitis, Huntington's disease, neuromuscular disorders, neuro-oncological diseases, neuro-immunological diseases, neuro-otological diseases, pain , Phobias, sleep disorders and other movement disorders. Other degenerative disorders include, for example, age related macular degeneration.

Target Nucleic Acid Non-coding RNA (ncRNA) concept: For more than half a century, central dogma in molecular biology has transcribed genetic information encoded in DNA to form an intermediate molecule, RNA, It was supposed to be translated into protein-forming amino acids. A promising hypothesis was that genes are directly related to proteins (“1 gene-1 protein”). In the last few years we have come to understand that the complexity of RNA levels is much more important than previously assumed. This complexity is mainly due to non-coding transcripts (as well as alternative splicing events), especially in eukaryotes (Mattick, JS (2004) RNA regulation: a new genetics? Nat Rev Genet 5 ( 4), 316-323).

  ncRNA contains microRNA, antisense transcripts, and other transcription units (TUs) that contain a high density of stop codons and lack any broad “open reading frame”. Many ncRNAs appear to begin at the start site in the 3 'untranslated region (3'UTR) of the protein coding locus. ncRNA is often rare and at least half of the ncRNAs sequenced by the FANTOM consortium do not appear to be polyadenylated. Most of the researchers have focused on polyadenylated mRNA that has been processed and transported into the cytoplasm for obvious reasons. Recently, it has been shown that a large number of non-polyadenylated nuclear RNAs can exist and many of these transcripts originate from so-called intergenic regions (Cheng, J. et al. (2005) Transcriptional maps of 10 human Sciences 308 (5725), 1149-1154; Kapranov, P. et al. (2005) Examples of the complex architecture of the human transcriptome revealed by RACE and high-density tiling arrays. Genome Res 15 ( 7), 987-997). See also FIG. 1A.

There is still some controversy about the concept of ncRNA, mainly because they share some but not all of the features of conventional coding RNA. Some ncRNAs appear to be highly conserved even in distantly related species. Most of the ncRNA analyzed by Carninci et al. (Carninci, P. et al. (2005) The transcriptional landscape of the mammalian genome. Science 309 (5740), 1559-1563) showed positional conservation between species. In considering function, transcription from a particular location may be important or may be the result of genomic structure or sequence. A non-coding transcript may in fact function through some sequence-specific interaction with the DNA sequence from which it is derived, or it may have other targets. Interestingly, the fact that ncRNA transcripts appear to evolve rapidly and they are often not well conserved does not necessarily mean that they lack function. There is a database of at least four web accessible ncRNAs (see Table 1).

  The best known mechanism by which ncRNA controls gene expression is through base pairing with the target transcript. An RNA that functions by base pairing is (1) a cis-encoded that is in the same genetic location as the RNA it acts on, but is encoded in the opposite strand and thus exhibits complete complementarity to its target. ) RNA, and (2) can be classified into trans-encoded RNAs that are encoded at a different chromosomal location than the RNA on which they act and generally do not show full base-pairing ability for their target .

  Antisense transcription: Antisense transcripts can be derived from coding RNA as well as non-coding RNA (FIG. 1A). Notably, antisense transcription in mammals is much more than could have been expected just a few years ago. Most recently, Katayama et al. (Katayama, S. et al. (2005) Antisense transcription in the mammalian transcriptome. Science 309 (5740), 1564-1566) reported that over 72% of all genome-mapped transcription units in mice. (43,553) showed overlap with some cDNA, 5 ′ or 3 ′ expressed sequence tag (EST) sequences, or tag or tag-pair regions mapped to opposite strands . At present, there is no reason to assume that this situation is very different in humans.

  However, it was argued that this transcriptional activity was mainly “unintentional” and “leakage” of the RNA transcription machinery. This latter proved that the antisense transcript pair is very likely to preserve its genomic organization through evolution compared to the non-antisense pair. Furthermore, expression analysis using strand-specific and conventional microarrays showed significant variation in the expression level of sense-antisense pairs among various mouse tissues. Large numbers of multiple sized transcripts are expressed from the sense-antisense locus, and they appear to lack a polyadenylation tail and tend to exhibit nuclear localization.

  There appear to be three basic types of sense-antisense pairs: (1) head-to-head or convergent, (2) tail-to-tail tail or divergent type, and (3) full overlap type. The divergent (head to head) type is most common (FIG. 1B).

  An important functional question is whether natural antisense pairs can form the basis of endogenous RNA interference (RNAi). Recent evidence showing that RNAi-based mechanisms are very widespread in different biological systems, and that in at least some species, part of the RNAi pathway can occur in the nuclear compartment, is antisense-mediated We suggest a possible role for this process in sexual gene inhibition. According to this aspect, double-stranded RNA (dsRNA) is cleaved into siRNA by Dicer or other RNase III family members. However, we recently demonstrated that the regulation of RNA by natural antisense transcripts occurs in human cells via a pathway independent of Dicer-related RNAi (Faghihi, M. and Wahlestedt, C. (2005 ) RNA interference is not involved in natural antisense mediated regulation of gene expression in mammals. Genome Biol). Antisense-sense interaction phenomena affect different types of genes and are heterogeneously distributed throughout the genome.

  RNA targeting: Drug discovery has historically focused on searching for compounds that regulate the protein product of a gene. Typically, these compounds are receptor agonists or antagonists, or compounds that inhibit or stimulate enzyme or protein-protein interactions. However, interest in targeting RNA for target validation and / or therapeutic purposes has increased, especially with the introduction of RNAi a few years ago. There are also many ongoing efforts aimed at targeting mRNA with small molecules, antisense oligonucleotides, ribozymes or aptamers.

  Current drug therapy involves only a few hundred endogenous targets, primarily receptors and enzymes. Genomics and transcriptomics efforts have identified a number of new drug target candidates that need to be validated. Target validation studies, such as RNA level manipulation, help to set priorities in the drug discovery process, but these do not directly lead to candidate drugs. Therefore, there are good reasons to continue to focus on established targets. Once they are found to be subject to natural antisense control, it will be possible to attempt to treat these “old” targets in new ways, especially when there are no drugs available for these targets .

  From the energetical point of view of genes encoding proteins, post-transcriptional control is an expensive mechanism for controlling gene expression. mRNA is only an intermediate in a multi-step process from gene to active protein. If the cell controls this process at the transcriptional level only at the beginning, it can save the energy required to provide, degrade, and recycle mRNA molecules that are not used to synthesize proteins. In order to modulate the action of ncRNA, RNA levels are clearly the only option for targeting.

  In a preferred embodiment, the oligonucleotide is targeted to the antisense and sense transcripts of the target nucleic acid. Oligonucleotides can be (1) head-to-head or divergent, (2) tail-to-tail or convergent, and (3) fully overlapping. FIG. 1B provides schematic diagrams of various types of orientations.

In another preferred embodiment, the oligonucleotide targets the antisense transcript ("mismatched control"), whereby knockdown of the antisense transcript alone increases expression of the traditional (sense) gene. Thus, in one aspect of the invention, expression of a desired gene can be upregulated. For example, if the target gene or nucleic acid encodes a known or putative drug target, knockdown of its antisense counterpart mimics the action of a receptor agonist or enzyme stimulator. Table 2 provides examples where knockdown of antisense (coding as well as non-coding) transcripts was shown to control sense expression inconsistently. For example, in Parkinson's disease, enhanced activity of a kinase localized in mitochondria, PINK1, (eg, increased PINK1) is desired:

  In a preferred embodiment, the siRNA molecule targets the overlap region of the desired sense / antisense locus, thereby controlling both sense and antisense transcripts.

  In preferred embodiments, the composition overlaps at the desired target locus and / or targets (overlaps or otherwise) both sense and antisense and one or more of the siRNA and A siRNA molecule of any combination of siRNA is included. These molecules can be directed to any target desired for possible treatment of any disease or disorder. For example, target cancer gene, receptor, gene encoding promoter, enhancer, cytokine and the like. Theoretically, there are no restrictions on which molecules are targeted. This invention pioneers a new era in the design of treatments for each disease and abnormality, whether congenital or not. Furthermore, the techniques taught herein allow treatment to be tailored to each individual.

  Another example in which upregulation of gene expression is desired by the compositions and methods of the present invention is in upregulation of angiogenesis, such as wound healing. To stimulate angiogenesis, enhanced signaling through the G protein-coupled receptor (GPCR), CD97, can be achieved by targeting its (code) antisense partner, Ddx-39.

  In another preferred embodiment, the oligonucleotide targets both the antisense and sense transcripts (“coincident control”), thereby knocking down the antisense and sense transcripts, and thereby the traditional (sense) gene. Achieve a synergistic reduction in expression. These concepts are illustrated in FIGS. 1A-1B and 2A-2B. When siRNA is used to achieve knockdown, this strategy overlaps one siRNA targeted to the sense transcript and another siRNA targeted to the corresponding antisense transcript, or simultaneously. It is further tested by applying a single energetically symmetrical siRNA that simultaneously targets the sense and antisense transcripts. According to Table 2, such dual simultaneous targeting is appropriate to pursue in the case of hypoxia-inducible factor 1 alpha, for example, a target whose inhibition can be beneficial in various disease states. Another example in Table 2 is the adrenomedullin AM1 receptor, a GPCR that has been shown that reduced signaling is also therapeutically beneficial.

Examples of compositions useful for the simultaneous knockdown of antisense and sense transcripts for use in Alzheimer's disease include, but are not limited to:

  These compositions are provided as illustrative examples and are not intended to be limiting. In preferred embodiments, the oligonucleotides can be tailored to individual treatments, for example, these oligonucleotides can be sequence specific to allelic variants in an individual, and target upregulation or inhibition can vary. Can be manipulated, for example, 10%, 20%, 40%, 100% expression relative to the control. That is, in some patients it may be effective to increase or decrease target gene expression by 10%, while in other patients it may be effective to increase or decrease 80%.

  Upregulation or inhibition of gene expression can be quantified by measuring endogenous target RNA or protein produced by translation of the target RNA. Techniques for quantifying RNA and proteins are well known to those skilled in the art. In some preferred embodiments, gene expression is inhibited by at least 10%, preferably at least 33%, more preferably at least 50%, even more preferably at least 80%. In particularly preferred embodiments of the invention, gene expression is inhibited in cells of the organism by at least 90%, more preferably at least 95%, or at least 99% to 100%. In some preferred embodiments, gene expression is upregulated by at least 10%, preferably at least 33%, more preferably at least 50%, even more preferably at least 80%. In particularly preferred embodiments of the invention, gene expression is upregulated in cells of the organism by at least 90%, more preferably at least 95%, or at least 99% to 100%.

  Selection of appropriate RNAi is facilitated by using computer programs that automatically align nucleic acid sequences and indicate regions of identity or homology. Such a program is used, for example, to compare nucleic acid sequences obtained by searching a database such as GenBank or nucleic acid sequences obtained by sequencing PCR products. Comparison of nucleic acid sequences from different species allows for the selection of nucleic acid sequences that exhibit an appropriate degree of identity between species. For genes that are not sequenced, Southern blots are performed to allow determination of the degree of identity between the gene in the target species and the gene of the other species. As is well known in the art, it is possible to obtain an approximate measure of identity by performing Southern blots with varying degrees of stringency. These procedures allow the selection of RNAi that exhibits high complementarity to the target nucleic acid sequence in the controlled subject and low complementarity to the corresponding nucleic acid sequence in other species. One skilled in the art understands that there is considerable freedom in selecting the appropriate region of a gene for use in the present invention.

  In a preferred embodiment, small interfering RNA (siRNA) is delivered to cells using RNA encoding sequences, either as RNA itself or as DNA, which encodes expression plasmids or small hairpin RNAs that are processed into viruses and siRNAs.

  In another preferred embodiment, the DNA cassette for cloning of the small hairpin sequence allows its expression and processing by RNA polymerase I, II or III. This system allows pre-siRNA to be efficiently transported into the cytoplasm where they are active, allowing the use of controlled and tissue specific promoters for gene expression.

  According to the present invention, any of the target cells, prokaryotes and eukaryotes is selectively targeted by siRNA based on their gene structure. Infectious diseases almost always result in the acquisition of foreign nucleic acids, which can be targeted using this technique. Specific targets may be viral, such as HIV (virus or provirus), or bacterial, such as multidrug resistant bacteria such as TB, fungi or protozoa. This technique may be particularly useful for the treatment of infections that lack an effective antimicrobial or antiviral agent (such as Ebola virus), or known or novel bioterrorism agents.

  Preferred siRNAs of the invention are targeted to target sequences, particularly target oligonucleotides for diseases such as Parkinson's disease, Alzheimer's disease, cancer, neurological diseases, infectious agents such as viral, bacterial, fungal or protozoan. Hybridize (bind) under stringency conditions, which can be assessed in vitro.

  The present invention can be used for gene products that encode proteins as well as gene products that do not encode proteins. Examples of gene products that do not encode proteins include gene products that encode RNA molecules involved in ribosomal RNA, transfer RNA, small nuclear RNA, cytoplasmic small RNA, telomerase RNA, DNA replication, chromosomal rearrangement, and the like.

  In another preferred embodiment, abnormal cells or cancer cells are targeted by siRNA. For example, many malignant tumors are accompanied by the presence of foreign DNA, such as Bcr-Abl, Bcl-2, HPV, which provide unique molecular targets to enable selective malignant cell targeting. . This approach can be used to target single base substitutions (eg K-ras, p53) or methylation changes. However, the growth of cancer cells can also be attributed to gene products that have not been previously expressed. These gene sequences can be targeted, thereby inhibiting further expression and resulting in ultimate death in cancer cells. In other examples, a transposon can be responsible for such deregulation and a transposon sequence, such as Tn5, can be targeted.

  According to the present invention, siRNA oligonucleotides are designed to be specific for molecules that cause, participate in, or exacerbate a disease state in a patient. For example, in viral infections, siRNA can be targeted to viral replication, viral infection cycles, eg, molecules responsible for adhesion to cellular ligands, viral gene products that encode host immune regulatory functions. In the present invention, particularly preferred viral organisms causing human diseases are (without limitation) filovirus, herpes virus, hepatitis virus, retrovirus, orthomyxovirus, paramyxovirus, togavirus, picornavirus, papovavirus, And gastroenteritis virus.

  In another preferred embodiment, the siRNA is targeted to a disease factor, such as a toxin produced by anthrax. For example, anthrax, which is one of the agents that can be used in a bioterror attack. Anthrax infection is mediated by Bacillus anthracis spores, which can enter the body through skin lacerations, through inhalation, or through feeding. Fatal anthrax is characterized by the establishment of systemic bacteremia with severe venemia. Anthrax appears to be a two-sided attack with bacteremia and / or venom that is involved in lethal syndromes of shock, hypoperfusion, and multisystem failure. The likelihood of developing systemic disease varies with the invading gate of the organism, and the route of inhalation is most prominent (reviewed in Dixon et al., 1999, New England J. Med. 341: 815-826 ). siRNA oligonucleotides may be targeted to mRNA that inhibits bacterial growth in infected patients, and may target toxin-producing gene products, thereby eliminating the toxic effects of Bacillus anthracis infection. Alternatively, siRNA can be targeted to any sequence target present in the organism and lacking in the host.

  The present invention generally relates to diseases resulting from diseases such as cancer and infectious agents such as viruses, intracellular and extracellular parasites, insertion factors, fungal infections, etc., and genes that are not normally expressed Provides a method for treating a disease that can cause aberrant expression, aberrant expression of a normally expressed gene or aberrant gene expression, which causes the disease to a patient in need of such treatment Administering an effective amount of an entity-specific siRNA oligonucleotide, or a mixture of various modified siRNAs, or a mixture of various modified and unmodified siRNAs.

  According to the present invention, siRNA oligonucleotide therapy involves the administration of siRNA oligonucleotides that contact (interact) with targeted mRNA derived from a gene, whereby the expression of the gene is controlled and expression is inhibited. . Such control of expression may suitably be at least about 10% or 20% different from the control, more preferably at least about 30%, 40%, 50%, 60%, 70%, 80 relative to the control. % Or 90% difference in expression. Interaction or contact with the siRNA oligonucleotide results in complete or substantially complete modulation of expression relative to the control, eg, inhibition of expression of at least about 95%, 97%, 98%, 99% or 100% relative to the control or It is particularly preferred to bring about an increase. A control sample for such modulation determination may be a corresponding cell (in vitro or in vivo) that has not been contacted with the siRNA oligonucleotide.

  The methods of the present invention provide for the treatment or prevention of diseases caused by abnormal cell proliferation and infectious agents, particularly the subject's lung, heart, liver, prostate, brain, testis, stomach, intestine, intestinal tract, spinal cord, sinuses. ), Preferably used for the treatment of infections that can occur in tissues such as the urinary tract or ovaries. The methods of the invention can also be used to treat systemic diseases such as viremia or sepsis. The methods of the present invention are also preferably used for the treatment of diseases and disorders associated with viral or bacterial infections, and any other disorder resulting from infectious agents.

  Preferably, the disease factor is isolated from the patient and identified using diagnostic tools such as ELISA, RIA, cell sorting, PCR and the like. However, the disease-causing factor may be a novel factor to which siRNA oligonucleotides can be targeted. Sequencing data obtained from the factors can be used to construct siRNA. Partial sequencing of factors can be achieved by any means known in the art. The following are provided as illustrative examples that are not intended to limit or interpret the present invention in any way. The siRNA is designed to be complementary to the selected sequence.

  According to one preferred embodiment of the invention, the nucleobase in the siRNA may be modified to provide greater specificity and affinity for the target mRNA. For example, nucleobases may be substituted with LNA monomers, which may be in contiguous stretches or at different positions. The modified siRNA preferably has a higher association constant (ka) than the complementary sequence relative to the target sequence. Binding of the modified or unmodified siRNA to the target sequence can be determined using a hybridization assay under various stringent conditions in vitro as described in the examples below.

  The basic properties of oligonucleotides that underlie many of its potential therapeutic applications are Watson-Crick hydrogen bonding (AT and GC) or other hydrogen bonding schemes, such as Hoogsteen / Reverse Hoog The ability to recognize and specifically hybridize to complementary single-stranded nucleic acids using the Stein mode or the like. Affinity and specificity are properties that are commonly used to characterize the hybridization properties of a particular oligonucleotide. Affinity is a measure of the strength of binding of an oligonucleotide to its complementary target (expressed as duplex thermal stability (Tm)). Each nucleobase pair in the duplex increases thermal stability, and thus affinity increases with increasing oligonucleotide size (number of nucleobases). Specificity is an indicator of the ability of an oligonucleotide to distinguish between perfectly complementary target sequences and mismatched target sequences. In other words, specificity is a measure of the loss of affinity associated with mismatched nucleobase pairs at the target.

  The usefulness of siRNA oligonucleotides for mRNA control (including inhibition) can be readily determined with simple tests. Thus, a specific siRNA oligonucleotide (modified or unmodified) is contacted with an in vitro or in vivo expression system containing the target mRNA, mutation or fragment thereof, and the level of expression is controlled, ie, siRNA oligo Comparisons can be made using the same expression system without contacting nucleotides.

  siRNA oligonucleotides may be used in combination. For example, a mixture of modified and / or unmodified different siRNA oligonucleotides directed to different regions of the same gene can be administered simultaneously or separately.

  In the practice of the present invention, the target gene product may be single-stranded or double-stranded DNA or RNA. A short dsRNA can be used to block transcription if this is the same sequence as the start site for transcription of a particular gene. See, for example, Janowski et al. Nature Chemical Biology, 2005, 10: 1038. It is understood that the targets to which the siRNA oligonucleotides of the invention are directed include the allelic form of the targeted gene and the corresponding mRNA containing splice variants. Given the knowledge of the sequence of the target polynucleotide, there is a lot of guidance in the literature for selecting specific sequences for siRNA oligonucleotides. Preferred mRNA targets include a 5 'cap site, a tRNA primer binding site, a start codon site, an mRNA donor splice site and an mRNA acceptor splice site.

  If the target polynucleotide comprises an mRNA transcript, the sequence-complementary oligonucleotide can hybridize to any desired portion of the transcript. Such oligonucleotides are, in principle, effective to inhibit translation and can induce the effects described herein. It is hypothesized that translation is most effectively inhibited by mRNA at or near the initiation codon. Accordingly, oligonucleotides complementary to the 5 'region of the mRNA transcript are preferred. Oligonucleotides complementary to the mRNA are preferred, including an initiation codon (the first codon at the 5 'end of the translated portion of the transcript) or a codon adjacent to the initiation codon.

Chimera / modified siRNA
In accordance with this invention, those skilled in the art will recognize not only the coding region in which the mRNA encodes the protein using a three letter genetic code including translation start and stop codons, but also the 5 ′ untranslated region, It is understood to include related ribonucleotides that form regions known as 3 ′ untranslated regions, 5 ′ cap regions, intron regions and intron / exon or splice site ribonucleotides and the like. Thus, the present invention can make oligonucleotides that are fully or partially targeted to these related ribonucleotides as well as the coding ribonucleotides. In preferred embodiments, the oligonucleotide is targeted to a translation initiation site (AUG codon) or sequence in the coding region, 5 ′ untranslated region or 3 untranslated region of the mRNA. The functions of the mRNA to be interfered with are all vital functions such as translocation of RNA to a site for protein translation, actual translation of protein from RNA, RNA splicing or maturation, and independence that may possibly involve RNA. Including even catalytic activity. The overall effect of such interference on RNA function is to cause interference with protein expression.

  Certain preferred oligonucleotides of this invention are chimeric oligonucleotides. A “chimeric oligonucleotide” or “chimera” is, in the context of this invention, an oligonucleotide comprising two or more chemically distinct regions each composed of at least one nucleotide. These oligonucleotides are typically modified nucleotides that confer one or more beneficial properties (eg, increased nuclease resistance, increased cellular uptake, increased binding affinity for RNA targets) And at least a region that serves as a substrate for an enzyme capable of cleaving RNA: DNA or RNA: RNA hybrids. As an example, RNase H is a cellular endonuclease that cleaves RNA strands of RNA: DNA duplexes. Activation of RNase H thus results in cleavage of the RNA target, thereby greatly enhancing the efficiency of antisense inhibition of gene expression. Thus, when using chimeric oligonucleotides, comparable results are often obtained with shorter oligonucleotides compared to phosphorothioate deoxyoligonucleotides that hybridize to the same target region. Cleavage of the RNA target can be detected as usual by gel electrophoresis and, if necessary, by related nucleic acid hybridization techniques known in the art. In one preferred embodiment, the chimeric oligonucleotide comprises at least one region modified to increase target binding affinity and a region that normally acts as a substrate for RNAse H. The affinity of an oligonucleotide for its target (in this case a nucleic acid encoding ras) is determined as usual by measuring the Tm of the oligonucleotide / target pair. The Tm of the oligonucleotide / target pair is the temperature at which the oligonucleotide and target dissociate, and dissociation is detected spectrophotometrically. The higher the Tm, the higher the affinity of the oligonucleotide for the target. In a more preferred embodiment, the region of the oligonucleotide to be modified comprises at least one nucleotide modified at the 2 ′ position of the sugar, most preferably 2′-O-alkyl, 2′-O-alkyl-O-alkyl or 2 '-Includes fluoro-modified nucleotides. In other preferred embodiments, the RNA modification comprises 2′-fluoro, 2′-amino and 2′O-methyl modifications, abasic residues in the ribose of pyrimidine, or an inverted base at the 3 ′ end of RNA. Including. Such modifications are incorporated into oligonucleotides as usual, and these oligonucleotides have been shown to have a higher Tm (ie higher target binding affinity) than 2'-deoxy oligonucleotides for a given target. ing. The effect of such increased affinity is to greatly enhance the inhibition of gene expression by RNAi oligonucleotides. RNase H is a cellular endonuclease that cleaves the RNA strand of RNA: DNA duplexes, and activation of this enzyme thus results in cleavage of the RNA target, thus greatly enhancing the efficiency of RNAi inhibition. Cleavage of the RNA target can be shown as usual by gel electrophoresis. In another preferred embodiment, the chimeric oligonucleotide is also modified to enhance nuclease resistance. Cells contain a variety of exonucleases and endonucleases that can degrade nucleic acids. Multiple nucleotide and nucleoside modifications have been shown to make oligodeoxynucleotides into which they are incorporated more resistant to nuclease degradation than native oligonucleotides. Nuclease resistance is routinely measured by incubating the oligonucleotide with a cell extract or isolated nuclease solution and measuring the extent of intact oligonucleotide remaining over time, usually by gel electrophoresis. The Oligonucleotides that have been modified to enhance their nuclease resistance will remain intact longer than unmodified oligonucleotides. Various oligonucleotide modifications have been shown to enhance or confer nuclease resistance. Oligonucleotides comprising at least one phosphorothioate modification are currently more preferred. In some cases, oligonucleotide modifications that increase target binding affinity can also independently enhance nuclease resistance. Several preferred modifications can be found in De Mesmaeker et al. Acc. Chem. Res. 1995, 28: 366-374.

Specific examples of multiple preferred oligonucleotides envisioned for this invention include modified backbones such as phosphorothioates, phosphotriesters, methylphosphonates, short alkyl or cycloalkyl intersugar linkages or short heteroatoms or heterocycles. Includes those containing a formula intersugar linkage. Most preferred are oligonucleotides having a phosphorothioate backbone, and heteroatom backbones, particularly CH ₂ —NH—O—CH ₂ , CH ₂ —N (CH ₃ ) —O—CH ₂ [methylene (methylimino) or as an MMI backbone _{known], (CH 2) -O-} N (CH 3) -CH 2, CH 2 -N (CH 3) -N (CH 3) -CH 2 and _{O-N (CH 3) -CH} 2 -CH An oligonucleotide with _two backbones (where the native phosphodiester backbone is represented as O—P—O—CH). The amide skeleton disclosed in De Mesmaeker et al. Acc. Chem. Res. 1995, 28: 366-374 is also preferred. Also preferred are oligonucleotides having a morpholino backbone structure (Summerton and Weller, US Pat. No. 5,034,506). In other preferred embodiments, such as a peptide nucleic acid (PNA) backbone, the phosphodiester backbone of the oligonucleotide is replaced with a polyamide backbone, and the nucleobase is bound directly or indirectly to the aza nitrogen atom of the polyamide backbone (Nielsen et al. Science 1991, 254, 1497). The oligonucleotide may also contain one or more substituted sugar residues. Preferred oligonucleotides contain one of the following at the 2 ′ position: OH, SH, SCH ₃ , F, OCN, OCH ₃ OCH ₃ , OCH ₃ O (CH ₂ ) _n CH ₃ , O (CH ₂ ) _n NH ₂ or O (CH ₂ ) _n CH ₃ (where n is 1 to about 10), C ₁ -C ₁₀ lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl, Cl, Br, CN, _{CF 3, OCF 3, O-,} S- or N- alkyl, O-, S- or, N- _{alkenyl, SOCH 3, SO 2 CH 3} , ONO 2, NO 2, N 3, NH 2, heterocycloalkyl , Heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving group, reporter group, intercalator A group for improving the pharmacokinetic properties of an oligonucleotide, or other substituents having a group for improving the pharmacodynamic properties of an oligonucleotide, and similar properties. A preferred modification includes 2'-methoxyethoxy _{[2'-OCH 2 CH 2 OCH} 3, also known as 2'-O (2-methoxyethyl)] containing (Martin et al., Helv. Chim. Acta, 1995, 78, 486). Other preferred modifications include 2'-methoxy _(2'-OCH 3), 2'- including propoxy _{(2'-OCH 2 CH 2 CH} 3) and 2'-fluoro (2'-F). Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3 ′ position of the sugar on the 3 ′ terminal nucleotide and the 5 ′ position of the 5 ′ terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl in place of the pentofuranosyl group.

Oligonucleotides may additionally or alternatively include modifications or substitutions of nucleobases (sometimes simply referred to in the art as simply “bases”). As used herein, “unmodified” or “natural” nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U). Modified nucleobases are nucleobases found rarely or only transiently in natural nucleic acids, such as hypoxanthine, 6-methyladenine, 5-Mepyrimidine, especially 5-methylcytosine (5-methyl-2′deoxycytosine Also referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentiobiosyl HMC, and synthetic nucleobases such as 2-aminoadenine, 2- (methylamino Adenine, 2- (imidazolylalkyl) adenine, 2- (aminoalkylamino) adenine or other hetero-substituted alkyladenine, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, _{7-deazaguanine,} N 6 (6- Ami Including hexyl) adenine and 2,6-diaminopurine). (Kornberg, A., DNA Replication, WH Freeman & Co., San Francisco, 1980, pp75-77; Gebeyehu, G., et al. Nucl. Acids Res. 1987, 15: 4513). “Universal” bases known in the art, such as inosine, may also be included. 5-Me-C substitutions have been shown to increase the stability of nucleic acid duplexes by 0.6-1.2 ° C (Sanghvi, YS, in Crooke, ST and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278).

  Another modification of the oligonucleotides of the invention involves chemically linking one or more moieties or conjugates that enhance the activity or cellular uptake of the oligonucleotide to the oligonucleotide. Such moieties include, without limitation, lipid moieties such as cholesterol moieties, cholesteryl moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA 1989, 86, 6553), cholic acid (Manoharan et al. Bioorg. Med). Chem. Let. 1994, 4, 1053), thioethers such as hexyl-S-tritylthiol (Manoharan et al. Ann. NY Acad. Sci. 1992, 660, 306, Manoharan et al. Bioorg. Med. Chem. Let. 1993, 3, 2765), thiocholesterol (Oberhauser et al., Nucl. Acids Res. 1992, 20, 533), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al. EMBO J. 1991 , 10, 111, Kabanov et al. FEBS Lett. 1990, 259, 327, Svinarchuk et al. Biochimie 1993, 75, 49), phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di -O-hexadecyl-rac-glycero-3-H-phosphonate (Mano haran et al. Tetrahedron Lett. 1995, 36, 3651, Shea et al. Nucl. Acids Res. 1990, 18, 3777), polyamine or polyethylene glycol chains (Manoharan et al. Nucleosides & Nucleotides 1995, 14, 969), or And adamantane acetic acid (Manoharan et al. Tetrahedron Lett. 1995, 36, 3651). Oligonucleotides containing lipophilic moieties and methods for preparing such oligonucleotides are known in the art (eg, US Pat. Nos. 5,138,045, 5,218,105 and 5,459,255).

  In a given oligonucleotide, it is not necessary for all positions to be uniformly modified, and indeed two or more of the modifications described above are single oligonucleotides or even single oligonucleotides within an oligonucleotide. It can be incorporated within a nucleoside. The invention also includes oligonucleotides that are chimeric oligonucleotides as defined above.

  In another embodiment, the nucleic acid molecule of the present invention is linked to another moiety, including but not limited to abasic nucleotides, polyethers, polyamines, polyamides, peptides, carbohydrates, lipids or polyhydrocarbon compounds. Also good. One skilled in the art will recognize that these molecules can be linked to one or more of any nucleotide, including nucleic acid molecules, at multiple positions on a sugar, base or phosphate group.

  The oligonucleotides used in this invention can be conveniently and routinely made by well-known techniques of solid phase synthesis. Equipment for such synthesis is sold by several vendors including Applied Biosystems. Any other means for such synthesis can also be used, and the actual synthesis of the oligonucleotide is well within the ability of one skilled in the art. It is also well known to use similar techniques to prepare other oligonucleotides such as phosphorothioates and alkylated derivatives. Using similar techniques and commercially available modified amidites and controlled pore glass (CPG) products such as biotin, fluorescein, acridine or psoralen modified amidites and / or CPG (available from Glen Research, Sterling VA) It is also well known to synthesize labeled, biotinylated, or other modified oligonucleotides, such as cholesterol-modified oligonucleotides.

  In accordance with the present invention, the use of modifications to enhance the potency, specificity and persistence of action of an oligonucleotide and to expand its route of administration, such as the use of LNA monomers, For example, MOE, ANA, FANA, PS, etc. are included (see Uhlman, Current Opinions in Drug Discovery & Development 2000 Vol 3 No 2). This can be achieved by replacing some of the monomers in the current oligonucleotide with LNA monomers. The LNA modified oligonucleotide may have a size similar to the parent compound, or may be larger or preferably smaller. Such LNA modified oligonucleotides comprise less than about 70%, more preferably less than about 60%, most preferably about 50% LNA monomer, and the size is about 10-25 nucleotides, more preferably about 12-20 nucleotides. It is preferable.

Cancer Treatment In another preferred embodiment, siRNA oligonucleotides are used in the treatment of patients who are susceptible to or developing cancer. Gene products that are overexpressed in cancer cells can be identified and siRNA oligonucleotides can selectively target cancer cells relative to normal cells. For example, an expression sequence tag (EST) can be used to identify nucleic acid molecules that are overexpressed in cancer cells [expression sequence tag (EST) sequencing (Celis, et al., FEBS Lett., 2000, Larsson, et al., J. Biotechnology., 2000, 80, 143-57] ESTs from various databases can be identified, for example, preferred databases include, for example, Online Mendelian Inheritance InMan (OMIM), Cancer Genome Anatomy Project (CGAP), GenBank, EMBL, PIR, SWISS-PROT, etc. OMIM, a database of genetic mutations related to diseases, is one of the National Center for Biotechnology Information. OMIM can be accessed via the Internet's World Wide Web, for example at ncbi.nlm.nih.gov/Omim/. GAP is an interdisciplinary program for establishing the information and technical tools necessary to elucidate the molecular dissection of cancer cells, such as ncbi.nlm.nih, via the Internet's world wide web. You can access them at .gov / ncicgap / Some of these databases may contain complete or partial nucleotide sequences, in addition to alternative transcript forms, private gene databases Alternatively, nucleic acid molecules can be selected from available publications or can be specifically determined for use in connection with the present invention.

  Selective transcription forms can be generated by computer software that generates contig sequences from individual ESTs in each of the databases. In another aspect of the invention, the nucleotide sequence of the target nucleic acid molecule is determined by combining multiple overlapping ESTs. An EST database (dbEST) known to and available to those skilled in the art includes about 1 million different human mRNA sequences composed of about 500-1000 nucleotides, and various numbers of ESTs from multiple different organisms. including. dbEST can be accessed via the Internet's world wide web, for example at ncbi.nlm.nih.gov/dbEST/index.html. These sequences are derived from a cloning strategy that uses cDNA expression clones for genome sequencing. ESTs are used to search for new gene products, map the genome, and identify coding regions in the genome sequence. Another important feature of EST sequence information that has become rapidly available is tissue-specific gene expression data. This can be extremely useful for targeting mRNA from one or more selective genes for therapeutic intervention. Since EST sequences are relatively short, they must be combined to provide a complete sequence. Since each available clone is sequenced, it results in multiple overlapping regions registered in the database. The net result is, for example, the derivation of selective transcript forms from normal and cancer cells.

  The combination of overlapping ESTs extending along both 5 'and 3' directions results in a full length "virtual transcript". The resulting virtual transcript may represent a nucleic acid that has already been characterized or may be a novel nucleic acid that does not have a known biological function. The Institute for Genomic Research (TIGR) Human Genome Index (HGI) database known to and available to those skilled in the art includes a list of human transcripts. TIGR can be accessed via the Internet's world wide web, for example at tigr.org. A transcript is an engine that builds a virtual transcript and can be generated in this way using a TIGR-Assembler known to those skilled in the art and available to those skilled in the art. TIGR-Assembler is a tool for combining large sets of overlapping sequence data, such as ESTs, BACs or small genomes, and can be used to combine eukaryotic or prokaryotic sequences. TIGR-Assembler is, for example, incorporated herein by reference and can be accessed via an Internet file transfer program, eg, tigr.org/pub/software/TIGR.assembler, for example, Sutton, et al., Genome Science & Tech., 1995, 1, 9-19. GLAXO-MRC, also known to and available to those skilled in the art, is another protocol for constructing virtual transcripts. The identification of ESTs and the generation of contig ESTs to form full-length RNA molecules are described in detail in US patent application Ser. No. 09/076440, which is incorporated herein by reference.

  Gene products that are overexpressed by cancer cells compared to normal cells can be identified, for example, gene products that are expressed at least 5 times more in pancreatic cancer than normal tissues. Gene expression can be analyzed by Serial Analysis of Gene Expression (SAGE) based on partial and defined sequence identification and characterization of transcripts corresponding to gene segments [SAGE (continuous analysis of gene expression) (Madden, et al., Drug Discov. Today, 2000, 5, 415-425)]. These defined transcript sequence “tags” are, for example, markers of gene products that are expressed in cells, tissues or extracts.

Identification of Target Nucleic Acid Sequence In a preferred embodiment, the inventive composition targets a desired nucleic acid sequence. The target nucleic acid sequence can be identified by various methods such as SAGE. SAGE is based on multiple principles. First, a short nucleotide sequence tag (9-10 bp) contains sufficient information content to uniquely identify a transcript if it is isolated from a defined position within the transcript. . For example, 9b. p. The short sequence of can distinguish 262,144 transcripts given a random nucleotide distribution at the tag site, while the human genome puts approximately 80,000-200,000 transcripts on putative It has been suggested to encode (Fields, et al., Nature Genetics, 7: 345 1994). The tag size may be shorter for lower eukaryotes or prokaryotes, for example, with fewer transcripts encoded by the genome. For example, 6-7b. p. This short tag would be sufficient to identify the transcript in yeast.

  Second, random dimerization of the tags allows for a procedure that reduces bias (due to amplification and / or cloning). Third, the ligation of these short sequence tags allows transcript continuous and efficient analysis by sequencing multiple tags within a single vector or clone. Similar to serial communication in a computer where information is sent as a continuation sequence of data, sequential analysis of array tags requires a means to establish registers and boundaries for each tag. The concept of obtaining a defined tag from a sequence according to the present invention is useful for matching a sample tag to a sequence database. In a preferred embodiment, computer techniques are used to match the sample sequence to the known sequence.

  The tag used here uniquely identifies the gene product. This is due to its length and its specific position (3 ') in the gene from which it is derived. Full-length gene products are identified by matching tags with genetic database components or by physically isolating previously unidentified gene products from cDNA libraries using tag sequences as probes be able to. Methods for isolating gene products from libraries using DNA probes are well known in the art. See, for example, Veculescu et al., Science 270: 484 (1995) and Sambrook et al. (1989), MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed. (Cold Spring Harbor Press, Cold Spring Harbor, NY). Once a gene or transcript is identified by matching with a database entry or by physically hybridizing to a cDNA molecule, the position of the hybridizing or matching region in the transcript can be determined. If the tag sequence is not at the 3 'end immediately adjacent to the restriction enzyme used to generate the SAGE tag, it is possible that a false match was made. Confirmation of the identity of the SAGE tag can be done by comparing the transcription level of the tag to that of the identified gene in a particular cell type.

  Analysis of gene expression is not limited to the methods described above and can include any method known in the art. All of these principles can be applied independently, in combination, or in combination with other known methods of sequence identification.

  Examples of gene expression analysis methods known in the art include DNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), READS (restriction enzyme amplification of digested cDNAs) (Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (total gene expression analysis (total gene expression analysis (Sutcliffe, et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 1976-81), protein arrays and proteomics (Celis, et al., FEBS Lett., 2000, 480, 2 -16, Jungblut, et al., Electrophoresis, 1999, 20, 2100-10), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal. Biochem., 2000, 286, 91-98, Larson , et al., Cytometry, 2000, 41, 203-208), subtractive cloning, differential display (DD) (Jurecic and Belmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomic hybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31, 286-96). ), FISH (fluorescence in situ hybridization) technique (Going and Gusterson, Eur. J. Cancer, 1999, 35, 1895-904), and mass spectrometry (Comb. Chem. High Throughput Screen, 2000, 3, 235). -41).

  In yet another aspect, siRNA oligonucleotides that selectively bind to variants of the target gene expression product are useful for the treatment of cancer. For example, p53 mutants are well known in various tumors. A “variant” is an alternative form of a gene. A variant may result from at least one mutation in the nucleic acid sequence and may result in altered mRNA or a polypeptide that may or may not be altered in structure or function. Any given natural or recombinant gene may not have an allelic form, or may have one or multiple allelic forms. Common mutational changes that give rise to variants are generally due to natural deletions, additions or substitutions of nucleotides. Each of these types of changes can occur alone or in combination with other changes in one or more places in a given sequence.

  Sequence similarity searches can be performed manually or using a number of available computer programs known to those skilled in the art. Preferably, the Blast and Smith-Waterman algorithms known to those skilled in the art and available to those skilled in the art can be used. Blast is NCBI's sequence similarity search tool designed to assist in the analysis of nucleotide and protein sequence databases. Blast can be accessed via, for example, ncbi.nlm.nih.gov/BLAST/ via the world wide web of the Internet. GCG Package provides a local version of Blast that can be used in a public domain database or with any locally available searchable database. GCG Package v9.0 is a commercially available software package that includes over 100 interrelated software programs that allow analysis by editing, mapping, comparing and aligning sequences. Other programs included in the GCG Package include, for example, programs that facilitate RNA secondary structure prediction, nucleic acid fragment assembly, and evolutionary analysis. In addition, the most prominent gene databases (GenBank, EMBL, PIR and SWISS-PROT) are distributed with the GCG Package and are fully accessible by database search and manipulation programs. GCG can be accessed via the Internet, for example, at http://www.gcg.com/. Fetch is a tool available in GCG that can get annotated GenBank records based on accession numbers and is similar to Entrez. Another sequence similarity search can be performed on GeneWorld and GeneThesaurus from Pangea. GeneWorld 2.5 is an automated, flexible, high-throughput application for polynucleotide and protein sequence analysis. GeneWorld enables automatic sequence analysis and annotation. Like GCG, GeneWorld incorporates multiple tools for homology search, gene search, multiple sequence alignment, secondary structure prediction and motif identification. GeneThesaurus 1.0 (TM) is a sequence and annotation data subscription service that provides information from multiple sources and provides a relational data model for public and local data.

  Another alternative sequence similarity search can be performed, for example, by BlastParse. BlastParse is a PERL script that runs on the UNIX platform that automates the strategies described above. BlastParse gets a list of targeted target accession numbers and parses all GenBank fields into “tab-delimited” text. This can then be saved in a “relational database” format for easier searching and analysis, providing flexibility. The end result is a series of fully parsed GenBank records that can be easily sorted, filtered and queried, as well as an annotation-relational database.

  According to the present invention, paralogs can be identified to design suitable siRNA oligonucleotides. A paralog is a gene in a species, which arises due to gene duplication, but has developed a new function and is also called an isotype.

  The polynucleotides of this invention can be isolated using the techniques described in the experimental section or replicated using PCR. PCR technology is the subject of US Pat. Nos. 4,683,195, 4,800,159, 4,754,065, and 4,683,202, PCR: The Polymerase Chain Reaction (Mullis et al. Eds, Birkhauser Press, Boston (1994)) and among them In the cited references. Alternatively, one of ordinary skill in the art can replicate DNA using the identified sequence and a commercially available DNA synthesizer. Thus, the present invention also provides a method for obtaining a polynucleotide of the present invention, which provides a linear sequence of the polynucleotide, nucleotides for its replication, appropriate primer molecules, chemicals such as enzymes and instructions. Provide a method by chemically replicating or ligating nucleotides in the appropriate orientation to obtain a polynucleotide. In another embodiment, these polynucleotides are further isolated. Still further, one of skill in the art can insert the polynucleotide into a suitable replication vector and insert the vector into a suitable host cell (prokaryotic or eukaryotic) for replication and amplification. The DNA amplified in this way can be isolated from the cells by methods well known to those skilled in the art. Here is further provided a method of obtaining a polynucleotide by this method, as well as the polynucleotide so obtained.

Disease Treatment In another preferred embodiment, siRNA can be used in the treatment of diseases in which immune cells are involved in the disease, such as autoimmune diseases, hypersensitivity to allergens, organ rejection, inflammation and the like. Examples of inflammation are associated with, for example, the following conditions: adult respiratory distress syndrome (ARDS) or multiple organ injury syndrome secondary to sepsis or trauma, myocardial or other tissue reperfusion injury, acute glomerulonephritis, Reactive arthritis, dermatoses due to acute inflammatory components, acute purulent meningitis or other central nervous system inflammatory diseases, burns, hemodialysis, leukocyte apheresis, ulcerative colitis, Crohn's disease, necrotizing enterocolitis, granulocyte transfusion Related syndromes and cytokine-induced toxicity. Examples of autoimmune diseases include, without limitation, psoriasis, type I diabetes, Raynaud's syndrome, autoimmune thyroiditis, EAE, multiple sclerosis, rheumatoid arthritis and lupus erythematosus.

  The methods of the invention can be used to screen for siRNA polynucleotides that inhibit the functional expression of one or more genes that control molecular expression associated with immunity. For example, the CD-18 family of molecules is important in cell adhesion. Due to the adhesion process, lymphocytes can always monitor the animal for the presence of foreign antigens. Although these processes are usually desirable, they are also responsible for transplant organ rejection, transplant tissue rejection and many autoimmune diseases. Therefore, siRNAs that can reduce or inhibit cell adhesion are highly desirable in organ transplant (eg, kidney transplant), tissue transplant recipients, or for autoimmune patients.

  In another preferred embodiment, the siRNA oligonucleotide inhibits the expression of MHC molecules involved in organ transplant or tissue transplant. For example, donor class I and class II molecules. siRNA inhibits the expression of these molecules, thereby improving the allograft response. Immune cells may be processed prior to organ or tissue transplantation and may be administered at the time of transplantation and / or any time thereafter, as may be determined by the attending physician. siRNA can be administered with or without immunosuppressant therapy.

  In another preferred embodiment, siRNA is used to treat individuals who are hypersensitive to antigen, such as allergic individuals. siRNAs are designed to target V region genes known to produce IgE molecules specific for allergens. IgE antibody specificity can be determined by routine immunodiagnostic techniques such as ELISA, RIA, PCR, Western blot, and the like. From the amino acid sequence of the IgE molecule, the nucleic acid sequence can be deduced using any of the database techniques described below. siRNAs are designed to bind to V region genes or any other part of the gene encoding the desired antibody, including rearranged and unrearranged immunoglobulin nucleic acid sequences.

  In another preferred embodiment, siRNAs are designed to target suppressor molecules that suppress the expression of genes that are not suppressed in normal individuals. For example, a suppressor molecule that inhibits a cell cycle-dependent gene, inhibition of p53 mRNA, inhibition of mRNA transcribed by a gene encoding a cell surface molecule, inhibition of caspases related to apoptosis and the like.

  Apoptosis is clinically important for several reasons. In the field of oncology, many clinically useful drugs kill tumor cells by inducing apoptosis. For example, cancer chemotherapeutic agents such as cisplatin, etoposide and taxol all induce apoptosis in target cells. In addition, various pathological disease states can arise from cells failing to properly control apoptosis. For example, failure of apoptosis can result in pathological accumulation of autoreactive lymphocytes such as occurs in many autoimmune diseases, and accumulation of virus-infected cells and hyperproliferative cells such as neoplasms It can also lead to accumulation of cells or tumor cells. SiRNAs that target mRNA from which proteins are translated and that can specifically induce apoptosis thus have therapeutic value in the treatment of these pathological disease states.

  On the other hand, inhibition of apoptosis is also clinically important. For example, cells are thought to die by apoptosis in the brain and heart after stroke and myocardial infarction, respectively. Furthermore, inappropriate activation of apoptosis can also cause various other pathological conditions including, for example, acquired immune deficiency syndrome (AIDS), neurodegenerative diseases and ischemic injury. As apoptotic inducers have been beneficial in the aforementioned disease states, specific inhibitors of apoptosis have therapeutic value in the treatment of these latter pathological disease states as well.

  In a preferred embodiment, the siRNA targets a normal expression of a therapeutically beneficial gene as described above, or optionally a gene that prevents overexpression. As used herein, the term “overexpressed”, when used in reference to the level of gene expression, means an increased accumulation of a gene product in an overexpressed cell compared to that level in a corresponding normal cell. And Overexpression can be achieved by natural biological phenomena, as well as by specific modifications, as in the case of genetically engineered cells. Overexpression also includes achieving an increase in the polypeptide that persists in the cell by an endogenous or exogenous mechanism. Overexpression due to natural phenomena can be caused, for example, by mutations that increase RNA expression, processing, transport, translation or stability, as well as mutations that result in increased stability or reduced degradation of the polypeptide. Examples of such increased expression levels are also examples of endogenous mechanisms of overexpression. A specific example of a natural biological phenomenon that results in overexpression by exogenous mechanisms is the close integration of retroviruses or transposons. Overexpression with certain modifications can be achieved, for example, by the use of siRNA oligonucleotides described herein.

  An siRNA polynucleotide can be constructed in a number of different ways, provided that it can interfere with the expression of the target protein. The siRNA polynucleotide is generally substantially identical to the target molecule sequence (although in a complementary direction). The minimum identity is typically about 80% or more, about 90% or more, about 95% or more, or about 100% identical.

Receptor Control and Candidate Therapeutic Agents In a preferred embodiment, cell surface receptors are controlled (controlled). Cell receptor regulation can be used, for example, for screening candidate drugs for disease treatment. Using siRNA, we provided experimental evidence that perturbation of antisense RNA by siRNA can alter the expression of the corresponding sense messenger RNA. However, this control can be consistent (antisense knockdown results in increased sense transcripts) or inconsistent (antisense knockdown results in reduced sense transcripts). Without wishing to be bound by theory, the concept of gene regulation is illustrated in FIGS. 1A-1B and 2A-2B. Table 2 shows various human and mouse antisense transcripts targeted by siRNA. In these cases, two or more siRNAs were targeted to the non-overlapping portion of the antisense strand and knockdown was confirmed using RT-PCR. Table 2 exemplifies the finding that coding antisense as well as non-coding antisense can be targeted in the same way, and the sense transcripts corresponding to either category can be controlled in a consistent or inconsistent manner. Here we propose two novel pharmacological strategies that may be based on knockdown of antisense RNA transcripts by siRNA (or other RNA targeting principles):

  Strategy 1: In the case of discordant control, the expression of a conventional (sense) gene can be increased by knocking down only the antisense transcript. If the latter gene encodes a known or putative drug target, knockdown of its antisense counterpart would be able to mimic the action of a receptor agonist or enzyme stimulant. Table 2 provides examples where knockdown of antisense (coding as well as non-coding) transcripts was shown to control sense expression inconsistently. For example, in Parkinson's disease, enhanced activity of a kinase localized in mitochondria, PINK1, (eg, increased PINK1) may be desirable, and knockdown of its non-coding antisense partner may be a means for that. Furthermore, in certain circumstances, to stimulate angiogenesis, enhanced signaling through the G protein-coupled receptor (GPCR), CD97, is due to targeting of its (code) antisense partner, Ddx-39. Can be achieved.

Strategy I example (increased PINK1, eg Parkinson's disease):

  Strategy 2: In the case of coordinated control, antisense and sense transcripts can be knocked down simultaneously, thereby achieving a synergistic reduction in conventional (sense) gene expression. These concepts are illustrated in FIGS. 2A and 2B. When siRNA is used to achieve knockdown, this strategy overlaps one siRNA targeted to the sense transcript and another siRNA targeted to the corresponding antisense transcript, or simultaneously. It is further tested by applying a single energetically symmetrical siRNA that simultaneously targets the sense and antisense transcripts. According to Table 2, such dual simultaneous targeting is appropriate to pursue in the case of hypoxia-inducible factor 1 alpha, for example, a target whose inhibition can be beneficial in various disease states. Another example in Table 2 is the adrenomedullin AM1 receptor, a GPCR that has been shown that reduced signaling is also therapeutically beneficial.

  With the advent of a new functional RNA world, there are new potential drug targets to consider. Among these are a number of naturally occurring antisense transcripts that have the ability to control the expression of sense transcripts, including those encoding conventional drug targets. Since many of these antisense transcripts are non-coding RNAs, they cannot be manipulated at the protein level. By using siRNA, we have shown that antisense transcript knockdown can result in either increased (consistent control) or decreased (mismatched control) sense transcript expression. These findings and concepts can form the basis for new pharmacological strategies.

  In a preferred embodiment, the method of identifying candidate therapeutics for the treatment of diseases such as Parkinson's disease, Alzheimer's disease, neurological disorders / diseases, tumors, etc. includes, for example, culturing cells (where cell receptors are inventions). For example, control (ie, upregulation or inhibition of receptor expression) and administration of a candidate therapeutic agent to cultured cells, the amount of receptor expression upon the presence or absence of the candidate therapeutic agent And associating phosphorylation relative to normal cells cultured in the presence of the candidate therapeutic and cells with controlled receptors, and drugs are identified based on the desired therapeutic outcome. As a result, for example, drugs that increase receptor expression, drugs that decrease receptor expression, drugs that phosphorylate or dephosphorylate the receptor, and the like are identified as candidate therapeutic agents that control the receptor.

  Another preferred method for diagnosis and candidate drug discovery includes contacting a test sample with a cell expressing a receptor or gene thereof, allele or fragment thereof, and the test sample and gene, allele or fragment thereof, Or detecting the interaction of the gene, its allele or fragment with the expression product. The desired gene, allele or fragment thereof, or the expression product of the gene, allele or fragment thereof is preferably detectably labeled, for example with a fluorescent or radioactive component.

  In another preferred embodiment, cells from a patient are isolated and contacted with a candidate therapeutic molecule. The gene, its expression product, is monitored to identify which gene or expression product is controlled by the drug. The identified gene controlled by the drug, an interfering RNA that controls the expression product can then be synthesized, thus providing a therapeutic oligonucleotide. These can be tailored to individual patients, which is advantageous because different patients do not respond equally effectively to the same drug. Thus, oligonucleotides provide individual treatment at a lower cost than conventional drug treatments.

  In one aspect, hybridization with an oligonucleotide probe capable of detecting a polynucleotide sequence, including a genomic sequence, encoding a desired gene or a closely related molecule is used to identify a target nucleic acid sequence. it can. The specificity of the probe depends on whether it was made from a very specific region, such as a 5 ′ regulatory region, or from a less specific region, such as a conserved motif, and hybridization or amplification (maximum Determine whether the probe can identify only genes, allelic variants or related sequences encoding naturally occurring sequences, regardless of (high, intermediate or low) stringency.

  The probe can also be used for the detection of related sequences, preferably at least 50% sequence identity to the sequence encoding any identified gene, more preferably any identified gene Should have at least about 60, 70, 75, 80, 85, 90 or 95 percent sequence identity (determining sequence identity is described above and Including use). The hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequences of the invention or from genomic sequences including gene promoters, enhancers and introns.

  As used herein, “homology” refers to subunit sequence similarity between two polymer molecules, eg, two nucleic acid molecules, such as two DNA molecules or two polypeptide molecules. If the positions of both subunits of two molecules are occupied by the same monomer subunit (eg, if a position of two DNA molecules is occupied by adenine), they are homologous at that position. is there. The homology between two sequences is a linear function of the number of matching positions or homologous positions. For example, in a sequence of two compounds, if 5 out of 10 positions are matched or homologous, the two sequences are 50% homologous and 9 out of 10 are matched or homologous In some cases, the two sequences have 90% homology. As an example, the DNA sequences 3'ATTGCC5 'and 3'TTTCCG5' have 50% homology.

Means for generating a specific hybridization probe for DNA encoding a target gene includes cloning a polynucleotide sequence encoding the target gene or derivative into a vector for generating an mRNA probe. Such vectors are known in the art and are commercially available and can be used to synthesize RNA probes in vitro by the addition of a suitable RNA polymerase and a suitable labeled nucleotide. Hybridization probes may be labeled with various reporter groups, for example with radionuclides such as ³² P or ³² S, or with enzyme labels such as alkaline phosphatase, fluorescent labels etc. bound to the probe by avidin-biotin binding systems. Can do.

  Polynucleotide sequences encoding target genes can be detected in Southern or Northern analysis, dot blots or other membrane-based techniques, in PCR techniques, in dipstick, pin and multiformat ELISA-like assays, and from patients or fluids or tissues Can be used in microarrays to detect altered target gene expression. Gel-based mobility shift analysis may be utilized. Other suitable qualitative or quantitative methods are well known in the art.

  The identity of a gene or variant thereof can be confirmed using techniques well known in the art. Examples include, without limitation, nucleic acid sequencing of amplified genes, hybridization techniques such as single nucleotide polymorphism analysis (SNP), and microarrays in which the molecules of interest are also immobilized on a biochip. Overlapping cDNA clones can be sequenced by dideoxy chain reaction using a fluorescent dye terminator and an ABI sequencer (Applied Biosystems, Foster City, Calif.). Any type of assay in which one component is immobilized can be performed using the substrate platform of the present invention. Bioassays that utilize immobilized components are well known in the art. Examples of assays using immobilized components include, for example, immunoassays, protein-protein interaction analysis, protein-nucleic acid interaction analysis, nucleic acid-nucleic acid interaction analysis, receptor binding assays, enzyme assays, phosphorylation assays, Includes diagnostic assays for determining disease status, gene profiling for drug compatibility analysis, SNP detection, and the like.

  Identification of a nucleic acid sequence that can bind to a target biomolecule is accomplished by immobilizing a library of nucleic acids on a substrate surface so that each unique nucleic acid is placed in a predetermined position to form an array. Can be achieved. The array is then exposed to the biomolecule under conditions that promote binding of the biomolecule to the nucleic acid. Non-specifically bound biomolecules can be washed away under mild to stringent buffer conditions, depending on the level of specificity of binding desired. The nucleic acid array is then analyzed to determine which nucleic acid sequence has bound to the biomolecule. Preferably, the biomolecule carries a fluorescent tag used to detect the position of the bound nucleic acid.

  Assays using fixed arrays of nucleic acid sequences include sequencing of unknown nucleic acids, single nucleotide polymorphism (SNP) analysis, analysis of gene expression patterns from specific species, tissues, cell types, etc., gene identification Can be used.

  Further diagnostic uses of oligonucleotides designed from sequences encoding the desired gene expression product may include the use of PCR. These oligomers may be chemically synthesized, produced enzymatically, or made in vitro. The oligomer preferably comprises a fragment of a polynucleotide encoding the expression product or a fragment of a polynucleotide complementary to the polynucleotide and is used under conditions optimized for the identification of a particular gene. Oligomers can also be used under conditions of lower stringency for the detection or quantification of closely related DNA or RNA sequences.

  In further embodiments, oligonucleotides or longer fragments from any polynucleotide sequence can be used as targets in a microarray. Microarrays monitor the identity and expression levels of multiple genes and gene transcripts while simultaneously identifying genes with which the target gene or product interacts and / or for example, the expression products of genes that mediate neurological diseases Can be used to evaluate the effectiveness of candidate therapeutics in control. This information can be used to determine gene function and to develop and monitor the activity of therapeutic agents.

  Microarrays can be prepared, used and analyzed using methods known in the art (Brennan et al., 1995, US Pat. No. 5,474,796, Schena et al., 1996, Proc. Natl. Acad. USA 93: 10614-10619, Baldeschweiler et al., 1995, PCT application WO95 / 251116, Shalon, et al., 1995, PCT application WO95 / 35505, Heller et al., 1997, Proc. Natl. Acad. Sci USA 94: 2150-2155 and Heller et al., 1997, US Pat. No. 5,605,662).

  Candidate agents include multiple chemical groups, but typically these are small organic compounds, nucleic acids including oligonucleotides, and organic compounds including peptides. The low molecular weight organic compound may have a molecular weight that is less, for example, preferably higher than about 40 or 50 but lower than about 2,500. Candidate agents may include functional chemical groups that interact with proteins and / or DNA.

  Candidate agents can be obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, a number of means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including the expression of randomized oligonucleotides. Alternatively, libraries of natural compounds, for example in the form of bacterial, fungal and animal extracts are available or are readily generated.

  The therapeutic agent assays of the present invention preferably include animal models, cell-based systems and non-cell-based systems.

  Preferably, the identified gene, variant, fragment or oligopeptide thereof is used to identify a therapeutically useful agent, eg, screening a library of compounds, or various drug screening Or by identifying the compound of interest by analytical techniques. The gene, its allele, fragment or oligopeptide used in such screening may be free in solution, attached to a solid support, supported on the cell surface, or intracellular May be located.

  Another technique for drug screening provides high-throughput screening of compounds with suitable binding affinity for the protein of interest (see, eg, Geysen et al., 1984, PCT application WO84 / 03564). In this method, a number of different small molecule test compounds are synthesized on a solid substrate. The test compound is reacted with the identified gene or fragment thereof and washed. The bound molecules are then detected by methods well known in the art. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.

  The screening methods of the present invention include using screening assays that identify one or more compounds having the desired activity from a library of diverse molecules. A “screening assay” is a selective assay designed to identify, isolate, and / or structure a compound having a preselected activity in a collection. “Identify” means that a compound having the desired activity is isolated and its chemical structure determined (without limitation, determining the respective nucleotide and amino acid sequences of nucleic acids and polypeptides, their structure, and , Additionally or alternatively, comprising purifying a compound having screened activity). Biochemical and biological assays are designed to test for activity in a wide range of systems ranging from protein-protein interactions, enzyme catalysis, small molecule-protein binding to cellular functions. Such assays include automated assays, semi-automated assays and HTS (High Throughput Screening) assays.

  In the HTS method, many separate compounds are preferably screened by robotic, automatic, or semi-automatic methods so that multiple test compounds are screened for the desired activity simultaneously or nearly simultaneously. Tested in parallel. By using the integrated system of the present invention, it is possible to assay and screen up to about 6,000 to 20,000 different compounds per day and even about 100,000 to 1,000,000 different compounds per day. Is possible.

  In HTS, the target molecule is typically administered to or cultured with isolated cells that have controlled receptors, including appropriate controls.

  In one embodiment, the screening involves contacting each cell culture with a diverse library of member compounds, some of which are target ligands, under conditions that allow target-ligand complexes to form. And identifying which members of the library are present in such complexes. In another non-limiting approach, the screening involves the target enzyme being converted into a diverse library of member compounds, some of which are target inhibitors or activators, the product or reaction of the reaction catalyzed by that enzyme. Contacting the object under conditions that produce a detectable signal. In the latter approach, the inhibitor of the target enzyme decreases the signal from the detectable product or increases the signal from the detectable reactant (and vice versa for the activator).

  Chemical libraries: Advances in combinatorial chemistry enable the rapid and economical synthesis of hundreds to thousands of separate compounds. These compounds are typically arranged in a medium size library of small molecules designed for efficient screening. Combinatorial methods can be used to generate unbiased libraries suitable for the identification of new compounds. It is also possible to create smaller, less diverse libraries derived from a single parent compound having a predetermined biological activity. In any case, the lack of effective screening systems that specifically target therapeutically relevant biomolecules produced by combinatorial chemistry, such as inhibitors of key enzymes, hinders the optimal use of these resources. Yes.

  A combinatorial chemical library is a collection of diverse chemical compounds created by combining multiple chemical “building blocks”, eg, reagents, either by chemical synthesis or biological synthesis. For example, a linear combinatorial chemical library, such as a polypeptide library, is a set of chemical building blocks (amino acids), in many combinations, and potentially in any possible way, for a given compound length (ie, in a polypeptide compound). The number of amino acids) in combination. Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.

  A “library” can contain from 2 to 50,000,000 diverse member compounds. Preferably, the library is at least 48 diverse compounds, preferably 96 or more diverse compounds, more preferably 384 or more diverse compounds, more preferably 10,000 or more diverse compounds, preferably 100,000 or more. And most preferably over 1,000,000 diverse member compounds. “Diverse” means that more than 50% of the compounds in the library have chemical structures that are not identical to any other member of the library. Preferably, more than 75%, more preferably more than 90%, and most preferably more than about 99% of the compounds in the library have a chemical structure that is not identical to any other member of the collection.

  The preparation of combinatorial chemical libraries is well known to those skilled in the art. For reviews, see Thompson et al., Synthesis and application of small molecule libraries, Chem Rev 96: 555-600, 1996, Kenan et al., Exploring molecular diversity with combinatorial shape libraries, Trends Biochem Sci 19: 57-64, 1994. , Janda, Tagged versus untagged libraries: methods for the generation and screening of combinatorial chemical libraries, Proc Natl Acad Sci USA. 91: 10779-85, 1994, Lebl et al., One-bead-one-structure combinatorial libraries, Biopolymers 37 : 177-98, 1995, Eichler et al., Peptide, peptidomimetic, and organic synthetic combinatorial libraries, Med Res Rev. 15: 481-96, 1995, Chabala, Solid-phase combinatorial chemistry and novel tagging methods for identifying leads, Curr Opin Biotechnol. 6: 632-9, 1995, Dolle, Discovery of enzyme inhibitors through combinatorial chemistry, Mol Divers. 2: 223-36, 1997, Fauchere et al., Peptide and nonpeptide lead discovery using robotically synthesized soluble libraries, Can J Physiol Pharmacol. 75: 683-9, 1997, Eichler et al., Generation and utilization of synthetic combinatorial libraries, Mol Med Today 1: 174-80, 1995, and Kay et al., Identification of enzyme inhibitors from phage-displayed combinatorial peptide libraries, Comb Chem High Throughput Screen 4: 535- See 43, 2001.

  Other chemical techniques for creating chemical diversity libraries can also be used. Such chemical techniques include, without limitation, peptoids (PCT publication WO 91/19735), encoded peptides (PCT publication WO 93/20242), random biooligomers (PCT publication WO 92/00091), benzodiazepines (US patents). No. 5,288,514), diversomers such as hydantoin, benzodiazepines and dipeptides (Hobbs, et al., Proc. Nat. Acad. Sci. USA, 90: 6909-6913 (1993)), vinyl-like polypeptides (Hagihara, et al , J. Amer. Chem. Soc. 114: 6568 (1992)), a non-peptidic peptidomimetic with a beta-D-glucose scaffold (Hirschmann, et al., J. Amer. Chem. Soc., 114). : 9217-9218 (1992)), similar organic synthesis of small molecule library (Chen, et al., J. Amer. Chem. Soc., 116: 2661 (1994)), oligocarbamate (Cho, et al. , Science, 261: 1303 (1993)) and / or peptidyl phosphonate (C ampbell, et al., J. Org. Chem. 59: 658 (1994)), nucleic acid library (see Ausubel, Berger and Sambrook above), peptide nucleic acid library (see, eg, US Pat. No. 5,539,083), antibody live Rally (see, eg, Vaughn, et al., Nature Biotechnology, 14 (3): 309-314 (1996) and PCT / US96 / 10287), carbohydrate libraries (eg, Liang, et al., Science, 274: 1520 -1522 (1996) and US Pat. No. 5,593,853), small organic molecule libraries (see, eg, benzodiazepines, Baum C & E News, January 18, page 33 (1993)), isoprenoids (US Pat. No. 5,569,588), thiazolidinones and Metathiazanone (US Pat. No. 5,549,974), pyrrolidine (US Pat. Nos. 5,525,735 and 5,519,134), morpholino compounds (US Pat. No. 5,506,337), benzodiazepines (US Pat. No. 5,288,514) and the like.

  Equipment for the preparation of combinatorial libraries is commercially available (eg, 357 MPS, 390 MPS, Advanced Chem. Tech, Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, Foster City, Calif. 9050 Plus, Millipore, Bedford, Mass.). A number of combinatorial libraries are also commercially available per se (eg, ComGenex, Princeton, NJ, Asinex, Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar, Ltd., Moscow, RU). 3D Pharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md., Etc.).

  High-throughput screening is a drug for complex molecular events such as signal transduction pathways and cell functions including, but not limited to, cell function, apoptosis, cell division, cell adhesion, migration, exocytosis and cell-cell communication It can be used to measure the action of Multicolor fluorescence allows multiple targets and cellular processes to be assayed on a single screen. The cross-correlation of cellular responses provides a wealth of information necessary for target evaluation and leads to optimization.

  In another aspect, the invention provides a method for analyzing cells, comprising a plurality of cells, wherein the cells provide an array of locations comprising one or more fluorescent reporter molecules, each location comprising a cell Scan multiple cells in to obtain fluorescent signals from intracellular fluorescent reporter molecules, convert fluorescent signals to digital data, and determine the distribution, environment or activity of fluorescent reporter molecules in cells That includes.

  A key component of the new drug discovery paradigm is the ever-increasing fluorescent and luminescent reagents used to measure the temporal and spatial distribution, content and activity of intracellular ions, metabolites, macromolecules and organelles A group. These reagent types measure labeling reagents that measure the distribution and amount of molecules in living and fixed cells, environmental indicators that report temporal and spatial signaling events, and measure target molecule activity in living cells. Including a fluorescent protein biosensor. A multi-parameter approach that combines multiple reagents into a single cell is a powerful new tool for drug discovery.

  This method relies on the high affinity of fluorescent or luminescent molecules for specific cellular components. Affinities for specific components include physical forces such as ionic interactions, covalent bonds (including chimeric fusions with protein-based chromophores, fluorophores and lumiphores), and hydrophobic interactions , Potential, and in some cases, controlled by simple capture within cellular components. Luminescent probes can be small molecules, labeled macromolecules or genetically engineered proteins, including but not limited to green fluorescent protein chimeras.

  Those skilled in the art will recognize a wide variety of fluorescent reporter molecules that can be used in the present invention, including but not limited to fluorescently labeled biomolecules such as proteins, phospholipids, RNA and DNA hybridization probes. . Similarly, specially synthesized fluorescent reagents with specific chemical binding or association properties have been used as fluorescent reporter molecules (Barak et al., (1997), J. Biol. Chem. 272: 27497). -27500, Southwick et al., (1990), Cytometry 11: 418-430, Tsien (1989) in Methods in Cell Biology, Vol. 29 Taylor and Wang (eds.), Pp. 127-156). Fluorescently labeled antibodies are particularly useful reporter molecules because of their high specificity for attaching to a single molecular target in a mixture of molecules as complex as a cell or tissue.

  Luminescent probes can be synthesized in living cells or cells in multiple non-mechanical modes including diffusion, facilitated or active transport, signal sequence mediated transport, and endocytotic or pinocytotic uptake Can be transported in. Mechanical bulk loading methods well known in the art can also be used to load luminescent probes onto living cells (Barber et al. (1996), Neuroscience Letters 207: 17-20, Bright et al. (1996), Cytometry 24: 226-233, McNeil (1989) in Methods in Cell Biology, Vol. 29, Taylor and Wang (eds.), Pp. 153-173). These methods include electroporation and other mechanical methods such as scrape loading, bead loading, impact loading, syringe loading, hypertonic loading and hypotonic loading. Also, as previously described, cells can be engineered to express a reporter molecule, such as GFP, bound to the protein of interest (Chalfie and Prasher US Pat. No. 5,491,084, Cubitt et al. al. (1995), Trends in Biochemical Science 20: 448-455).

Within the cell, the luminescent probe accumulates in the target region as a result of specific high affinity interactions with the target region or other modes of molecular targeting, such as signal sequence mediated transport. Fluorescently labeled reporter molecules are useful for determining the location, amount and chemical environment of the reporter. For example, it can be determined whether the reporter is in a lipophilic membrane environment or in a more aqueous environment (Giuliano et al. (1995), Ann. Rev. of Biophysics and Biomolecular Structure 24: 405 -434, Giuliano and Taylor (1995), Methods in Neuroscience 27.1-16). The pH environment of the reporter can be determined (Bright et al. (1989), J. Cell Biology 104: 1019-1033, Giuliano et al. (1987), Anal. Biochem. 167: 362-371, Thomas et al. (1979), Biochemistry 18: 2210-2218). It can be determined whether a reporter having a chelating group is bound to an ion, such as Ca ⁺⁺ (Bright et al. (1989), in Methods in Cell Biology, Vol. 30, Taylor and Wang (eds.), pp. 157-192, Shimoura et al. (1988), J. of Biochemistry (Tokyo) 251: 405-410, Tsien (1989) in Methods in Cell Biology, Vol. 30, Taylor and Wang (eds.), pp. 127-156).

  Furthermore, some cell types in the organism may contain components that can be specifically labeled that may not be present in other cell types. For example, nerve cells often contain polarized membrane components. That is, these cells distribute macromolecules asymmetrically along their plasma membrane. Connective tissue cells or supportive tissue cells often contain granules in which molecules specific to that cell type are trapped (eg, heparin, histamine, serotonin, etc.). Most muscle tissue cells contain sarcoplasmic reticulum, a specialized organelle that functions to control the concentration of calcium ions in the cytoplasm. Many neural tissue cells contain secretory granules and vesicles in which are trapped neurohormones or neurotransmitters. Thus, fluorescent molecules can be designed to label not only specific components within specific cells, but also specific cells within a population of diverse cell types.

  Those skilled in the art are aware of a wide variety of methods for measuring fluorescence. For example, certain fluorescent reporter molecules exhibit a change in excitation or emission spectrum, some are resonant energy transfer (where one fluorescent reporter loses fluorescence while the other increases fluorescence). Some species show the disappearance (quenching) or appearance of fluorescence, while others report rotational motion (Giuliano et al. (1995), Ann. Rev. of Biophysics and Biomol. Structure). 24: 405-434, Giuliano et al. (1995), Methods in Neuroscience 27: 1-16).

  The entire procedure can be fully automated. For example, sampling of sample material involves taking a sample from a sample container and delivering at least a portion of the collected sample to a test cell culture (eg, a cell culture in which gene expression is controlled). It can be achieved by a plurality of steps including. Sampling may also include additional steps, particularly and preferably a sample preparation step. In one approach, only one sample at a time is taken on the autosampler probe and only one sample is present on the probe at a time. In another embodiment, multiple samples can be collected on autosampler probes separated by a solvent. In yet another aspect, multiple probes can be used in parallel for automatic sampling.

  In the general case, sampling can be done manually, in a semi-automatic manner, or in an automatic manner. The sample can be taken manually from the sample container, eg, with a pipette or with a syringe-type manual probe, and then manually delivered to the loading port or injection port of the characterization system. In semi-automated protocols, some aspects of the protocol occur automatically (eg, delivery), while some other aspects require manual interference (eg, sample collection from the process control line). Preferably, however, the sample or samples are taken from the sample container and delivered to the characterization system in a fully automated manner, for example by an autosampler.

  In one aspect, automatic sampling may be done using a microprocessor that controls an automation system (eg, a robot arm). Preferably, the microprocessor has a library of samples with various arrangements of samples (eg, "n rows" x "n columns" square array, "n rows" x "m columns" rectangular array, circular array, “R” × “r” × “r” equilateral triangle array, “r base” × “s” × “s” isosceles triangle array, etc. where n, m, r and s are Can be programmed by the user to support (which is an integer).

  Automated sampling of sample material can optionally be performed with an autosampler having a heated injection probe (chip). An example of such an autosampler is disclosed in US Pat. No. 6,175,409 B1 (incorporated by reference).

  According to the present invention, one or more systems, methods or both are used to identify a plurality of sample materials. Although manual or semi-automated systems and methods are possible, preferably automated systems or methods are used. Various robotic systems or automated systems perform predetermined actions for handling, contacting, dispensing or otherwise manipulating materials in the form of solids, liquid fluids or gases according to predetermined protocols. Available to provide, automatically or programmably. Such a system can be adapted or augmented to include various hardware, software, or both to assist the system in determining the mechanical properties of the material. Hardware and software for augmenting the robotic system includes, without limitation, sensors, transducers, data acquisition and manipulation hardware, data acquisition and manipulation software, and the like. Typical robotic systems are commercially available from CAVRO Scientific Instruments (eg, Model NO.RSP9652) or BioDot (Microdrop Model 3000).

  Typically, automated systems have appropriate protocol design and execution software that can be programmed with information, such as synthesis, composition, location information, or other information related to a library of materials placed over a substrate. Including. Protocol design and execution software typically communicates with robot control software to control a robot or other automated device or system. The protocol design and execution software also communicates with data acquisition hardware or software for the software to collect data from the reaction measurement hardware. Once the data is collected in the database, analysis software can be used to analyze the data, and more specifically to determine the characteristics of the candidate drug, or the data can be analyzed manually. it can.

  In another preferred embodiment, assaying a candidate drug or sample by cell culture is combined with one or more methods. In one embodiment, the sample can be pre-fractionated according to the size of the protein in the sample using size exclusion chromatography. For biological samples where the amount of sample available is small, preferably a size selective spin column is used. In general, the first fraction eluting from the column (“Fraction 1”) has the highest percentage of high molecular weight protein, fraction 2 has the lower percentage of high molecular weight protein, and fraction 3 has , With a lower percentage of high molecular weight protein, fraction 4 has the lowest amount of large protein. Each fraction can then be analyzed for detection of the compound by immunoassay, gas phase ion spectrometry, and the like.

  In another embodiment, the sample can be pre-fractionated by anion exchange chromatography. Anion exchange chromatography allows proteins in a sample to be pre-fractionated roughly by their charge characteristics. For example, a Q anion exchange resin can be used (eg, Q HyperD F, Biosepra) and samples can be eluted sequentially with eluents having different pHs. Anion exchange chromatography allows the separation of compounds in samples that are more negatively charged than other types of compounds. Proteins eluted with an eluent having a high pH are considered slightly negatively charged, and fractions eluted with an eluent having a low pH are considered strongly negatively charged. Thus, in addition to reducing sample complexity, anion exchange chromatography separates proteins by their binding characteristics.

  In yet another embodiment, the sample can be pre-fractionated by heparin chromatography. Heparin chromatography also allows pre-fractionation of compounds in a sample based on affinity interactions with heparin and charge characteristics. Heparin, a sulfated mucopolysaccharide, binds compounds with positively charged moieties, and samples can be eluted sequentially with eluents having different pH or salt concentrations. Samples that elute with an eluent having a low pH are considered to be slightly negatively charged. Samples eluted with an eluent having a high pH are considered to be strongly negatively charged. Thus, anion exchange chromatography also separates proteins by their binding characteristics, in addition to reducing sample complexity.

  In yet another embodiment, the sample can be pre-fractionated by isolating proteins with specific characteristics, such as glycosylated proteins. For example, a CSF sample can be fractionated by passing the sample through a lectin chromatography column, which has a high affinity for sugars. Glycosylated proteins bind to the lectin column and non-glycosylated proteins pass through the flow-through. The glycosylated protein is then eluted from the lectin column with an eluent containing a sugar, such as N-acetyl-glucosamine, and is available for further analysis.

  Thus, there are many ways to reduce the complexity of a sample based on the binding properties of the protein in the sample or the characteristics of the protein in the sample.

Delivery of siRNA The practice of the preferred invention involves administering at least one of the aforementioned siRNA polynucleotides in a suitable nucleic acid delivery system. In one embodiment, the system includes a non-viral vector operably linked to the polynucleotide. Examples of such non-viral vectors include polynucleosides, alone or in combination with suitable protein, polysaccharide or lipid formulations.

  Further suitable nucleic acid delivery systems are from viral vectors, typically at least one of adenovirus, adenovirus-associated virus (AAV), helper-dependent adenovirus, retrovirus or Sendai virus (HVJ) -liposome complex. Contains an array. Preferably, the viral vector comprises a strong eukaryotic promoter, such as a cytomegalovirus (CMV) promoter, operably linked to the polynucleotide.

  Further preferred vectors include viral vectors, fusion proteins and chemical conjugates. Retroviral vectors include Moloney murine leukemia virus and HIV-based viruses. One preferred HIV-based viral vector comprises at least two vectors in which the gag and pol genes are from the HIV genome and the env gene is from another virus. DNA viral vectors are preferred. These vectors include pox vectors such as orthopox vectors or avipox vectors, herpes virus vectors such as herpes simplex virus type I (HSV) vectors [Geller, AI et al., J. Neurochem, 64: 487 ( 1995), Lim, F., et al., In DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England) (1995), Geller, AI et al., Proc Natl. Acad. Sci .: USA: 90 7603 (1993), Geller, AI, et al., Proc Natl. Acad. Sci USA: 87: 1149 (1990)], adenoviral vector [LeGal LaSalle et al., Science, 259: 988 (1993), Davidson, et al., Nat. Genet. 3: 219 (1993), Yang, et al., J. Virol. 69: 2004 (1995)] and adeno-associated virus vectors [Kaplitt, MG, et al ., Nat. Genet. 8: 148 (1994)].

  A poxvirus vector introduces a gene into the cytoplasm. Avipox virus vectors provide only short term expression of nucleic acids. Adenovirus vectors, adeno-associated virus vectors, and herpes simplex virus (HSV) vectors may be suitable for multiple inventive aspects. Adenoviral vectors provide shorter term expression (eg, less than about 1 month) than adeno-associated viruses, and in some embodiments may exhibit longer expression. The particular vector chosen will depend on the target cell and the condition being treated. Selection of an appropriate promoter can be easily achieved. Preferably, a high expression promoter can be used. An example of a suitable promoter is the 763 base pair cytomegalovirus (CMV) promoter. Rous sarcoma virus (RSV) (Davis, et al., Hum Gene Ther 4: 151 (1993)) and the MMT promoter can also be used. A protein can be expressed using its native promoter. Other elements that can enhance expression may also include enhancers or systems that provide high levels of expression, such as the tat gene and tar elements. This cassette can then be inserted into a vector, eg, a plasmid vector such as pUC19, pUC118, pBR322, or other known vector containing, for example, an E. coli origin of replication. See, for example, Sambrook, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory press, (1989). The plasmid vector may also be a selectable marker, eg, a β-lactamase gene for ampicillin resistance, provided that the marker polypeptide does not adversely affect the metabolism of the treated organism. The cassette can also be bound to a nucleic acid binding moiety in a synthetic delivery system, such as the system disclosed in WO 95/22618.

  If desired, the polynucleotides of the invention can also be used with microdelivery vehicles such as cationic liposomes and adenoviral vectors. For a review of procedures for liposome preparation, targeting, and delivery of contents, see Mannino and Gould-Fogerite, BioTechniques, 6: 682 (1988). See also Felgner and Holm, Bethesda Res. Lab. Focus, 11 (2): 21 (1989) and Maurer, R.A., Bethesda Res. Lab. Focus, 11 (2): 25 (1989).

  Replication-deficient recombinant adenoviral vectors can be produced by known techniques. Quantin, et al., Proc. Natl. Acad. Sci. USA, 89: 2581-2584 (1992), Stratford-Perricadet, et al., J. Clin. Invest., 90: 626-630 (1992), and See Rosenfeld, et al., Cell, 68: 143-155 (1992).

  Another preferred siRNA delivery method is to use a single-stranded DNA production vector that can produce siRNA intracellularly. See, for example, Chen et al, BioTechniques, 34: 167-171 (2003), which is incorporated herein by reference in its entirety.

  The effective amount of nucleic acid depends on the specific expressed protein, the specific cardiac arrhythmia targeted, the patient and its clinical condition, weight, age, sex, and the like.

  One preferred delivery system is a recombinant viral vector that incorporates one or more polynucleotides therein, preferably about one polynucleotide. Preferably, the viral vector used in the method of the invention has a pfu (plaque forming unit) from about 108 to about 5 × 10 10 pfu. In embodiments where the polynucleotide is administered by a non-viral vector, it is often useful to use about 0.1 nanogram to about 4000 micrograms, such as about 1 nanogram to about 100 micrograms.

Assessment of up-regulation or inhibition of gene expression Transfer by an exogenous nucleic acid vector into a host cell or organism can be assessed by detecting the presence of the nucleic acid directly in the cell or organism. Such detection can be accomplished by a number of methods well known in the art. For example, the presence of exogenous nucleic acid can be detected by Southern blot or by polymerase chain reaction (PCR) techniques using primers that specifically amplify the nucleotide sequence associated with the nucleic acid. The expression of exogenous nucleic acid can also be measured using conventional methods. For example, mRNA produced from exogenous nucleic acid can be detected and quantified using Northern blot and reverse transcription PCR (RT-PCR).

  Expression of RNA from the exogenous nucleic acid can also be detected by measuring enzyme activity or reporter protein activity. For example, siRNA activity can be measured indirectly as a reduction or increase in target nucleic acid expression, which is a measure of how exogenous nucleic acid is producing effector RNA. Based on sequence conservation, primers can be designed and used to amplify the coding region of the target gene. Initially, the most highly expressed coding region from each gene can be used to construct a model control gene, although any coding region or non-coding can be used. Each control gene is assembled by inserting each coding region between the reporter coding region and its poly (A) signal. These plasmids produce a reporter gene in the upstream part of the gene and an mRNA with a potential RNAi target in the 3 'non-coding region. The effect of individual RNAi is assessed by changes in the reporter gene. Reporter genes useful in the methods of the present invention include acetohydroxyacid synthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucuronidase (GUS), chloramphenicol acetyltransferase (CAT), green Fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopine synthase ( OCS) and derivatives thereof. Multiple selectable markers are available that confer resistance to ampicillin, bleomycin, chloramphenicol, gentamicin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin and tetracycline. Methods for determining reporter gene changes are well known in the art and include, but are not limited to, fluorescence analysis methods (eg, fluorescence spectroscopy, fluorescence activated cell sorting (FACS), fluorescence microscopy), antibiotics Includes determination of substance resistance.

  Biological genome information and model genes are invaluable for high-throughput screening of potential RNAi, but interference activity against target nucleic acids must ultimately be demonstrated experimentally in cells expressing the target nucleic acid Don't be. To determine the ability of an RNAi sequence to interfere, an RNAi-containing vector is transfected into an appropriate cell line that expresses the target nucleic acid. Each selected RNAi construct is tested for its ability to alter the steady state mRNA of the target nucleic acid. In addition, any target mRNA that “survived” in the first round of testing is amplified and sequenced by reverse transcription-PCR (eg, Sambrook, J. et al. “Molecular Cloning: A Laboratory Manual,” 2nd addition , Cold Spring Harbor Laboratory Press, Plainview, NY (1989)). These sequences are analyzed to determine individual polymorphisms that allow mRNA to escape the current RNAi library. This information is also used to further modify the RNAi construct to target even rarer polymorphisms.

  Methods for transfecting cells with RNAi vectors are well known in the art and include, without limitation, electroporation, particle bombardment, microinjection, viral vector transfection, retroviral based vector transfection. And liposome-mediated transfection. Any type of nucleic acid that mediates RNA interference can be synthesized in vitro and inserted directly into cells using a variety of methods well known in the art. DsRNA and other molecules that mediate RNA interference are also available from commercial vendors such as Ribopharma AG (Kulmach, Germany), Eurogentec (Seraing, Belgium), Sequitur (Natick, Mass.) And Invitrogen (Carlsbad, Calif.). Etc. are available. Eurogentec is labeled with a fluorophore (eg, HEX / TET; 5'-fluorescein, 6-FAM; 3'-fluorescein, 6-FAM; fluorescein dT internal; 5 'TAMRA, rhodamine; 3' TAMRA, rhodamine) DsRNA are also provided and can also be used in the present invention. RNAi molecules can be made by well-known solid phase synthesis techniques. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Other methods for such synthesis known in the art can additionally or alternatively be used. It is well known to use similar techniques to prepare oligonucleotides, such as phosphorothioates and alkylated derivatives.

RNA directly inserted into cells may contain modifications in the phosphate-sugar backbone or nucleoside. For example, the phosphodiester bond of natural RNA can be modified to include at least one of nitrogen or sulfur heteroatoms. Interfering RNA can be produced enzymatically or by partial / complete organic synthesis. The construct can be synthesized by cellular RNA bacteriophage or bacteriophage RNA polymerase (eg, T3, T7, SP6). When synthesized chemically or by in vitro enzymatic synthesis, RNA may be purified prior to introduction into cells or animals. For example, RNA can be purified from a mixture by extraction with a solvent or resin, precipitation, electrophoresis, chromatography, or a combination thereof, as is known in the art. Alternatively, interfering RNA constructs can be used without purification or with minimal purification to avoid loss due to sample processing. The RNAi construct can be dried for storage or can be dissolved in an aqueous solution. The solution may contain buffers or salts to facilitate duplex annealing and / or stabilization. Examples of buffers or salts that can be used in the present invention include, but are not limited to, saline, PBS, N- (2-hydroxyethyl) piperazine-N ′-(2-ethanesulfonic acid) (HEPES ™). ), 3- (N-morpholino) propanesulfonic acid (MOPS), 2-bis (2-hydroxyethylene) amino-2- (hydroxymethyl) -1,3-propanediol (bis-TRIS ™), phosphorus Potassium phosphate (KP), sodium phosphate (NaP), disodium hydrogen phosphate (Na ₂ HPO ₄ ), sodium dihydrogen phosphate (NaH ₂ PO ₄ ), potassium potassium phosphate (NaKHPO ₄ ), magnesium phosphate ( _{mg 3 (PO 4) 2 -4H} 2 O), potassium acetate _{(CH 3 COOH), D (} +) - α- sodium glycerophosphate ( _{HOCH 2 CH (OH) CH 2} OPO 3 Na 2), and includes other physiologic buffers known to those skilled in the art. Further buffers used in the present invention include salt MX dissolved in an aqueous solution, its associated or dissociated product, where M is an alkali metal (eg, Li ⁺ , Na ⁺ , K ⁺ , Rb ⁺ ), preferably Wherein X is an anion, the salt comprising an anion selected from the group consisting of phosphate, acetate, bicarbonate, sulfate, pyruvate, and an organic monophosphate. Glucose 6-phosphate or DL-α-glycerol phosphate.

Genes Controlled / Targeted by RNAi Molecules In a further aspect of the invention, RNAi molecules that control the expression of specific genes or gene families are provided, so that gene expression can be functionally deleted or upregulated. . In one embodiment, at least two RNAi molecules that target the same region of the gene are provided. In another aspect, at least two RNAi molecules are provided that target at least two different regions of the same gene. In a further aspect, at least two RNAi molecules targeting at least two different genes are provided. A further aspect of the invention provides a combination of the above strategies for gene targeting.

  In one embodiment, the RNAi molecules may be the same sequence. In alternative embodiments, the RNAi molecule may be a different sequence. In other embodiments, at least two RNAi molecules are provided, wherein one or more families of genes can be controlled by expression of the RNAi molecule. In another aspect, at least three, four, or five RNAi molecules are provided, wherein one or more families of genes can be controlled by expression of the RNAi molecule. The RNAi molecule may be homologous to a conserved sequence in one or more genes. The family of genes controlled using such a method of the invention includes those endogenous to the cell, related viral gene families, conserved gene families in viral genera, related eukaryotic parasite genes Or more specifically a family of genes from porcine endogenous retroviruses. In one particular embodiment, the at least two RNAi molecules can target at least two different genes that are members of the same gene family. RNAi molecules can target homologous regions in a family of genes, and thus one RNAi molecule can target the same region of multiple genes.

  The RNAi molecule can be selected from, but not limited to, the following types of RNAi: antisense oligonucleotide, ribozyme, small interfering RNA (sRNAi), double stranded RNA (dsRNA), inverted repeat, short chain Hairpin RNA (shRNA), small temporally regulated RNA, and radial clustered inhibitory RNA, asymmetric clustering inhibitory RNA, linear clustering inhibitory RNA and complex or Clustering inhibitory RNA (cRNAi), including complex or compound clustering inhibitory RNA.

  In another aspect, the expression of the RNAi molecule to control the target gene in a mammalian cell line or transgenic animal is such that the expression of the target gene is functionally deleted or is below a detectable level, Or provided to be upregulated, ie, the expression of the target gene is reduced or increased by at least about 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99%.

  In another embodiment of this aspect of the invention, methods are provided for producing cells and animals in which interfering RNA molecules are expressed to control the expression of target genes. For example, the method according to this aspect of the invention may include, for example: identifying one or more target nucleic acid sequences in a cell, obtaining at least one RNAi molecule that binds to one or more target nucleic acid sequences, optionally expressing Introducing an RNAi molecule packaged in a vector into a cell, and under conditions where RNAi binds to a target nucleic acid sequence in the cell, thereby controlling expression of one or more target genes Expression.

  In an embodiment of the present invention, the endogenous gene that can be controlled by the expression of at least one RNAi molecule is not limited, but is a gene required for cell survival or cell replication, a gene required for virus replication, a gene immunoglobulin gene Genes encoding loci, such as kappa light chains, and genes encoding cell surface proteins, such as vascular cell adhesion molecule (VCAM), and other genes important for the structure and / or function of cells, tissues, organs and animals including. The methods of the invention can also be used to control the expression of one or more non-coding RNA sequences. These non-coding RNA sequences may be RNA viral genomes, endogenous genes, eukaryotic parasite gene sequences, or other non-coding RNA sequences known in the art and familiar to those skilled in the art. May be. In accordance with aspects of the invention, an RNAi molecule expressed in a cell or animal can reduce, increase or maintain the expression of one or more target genes. To identify specific target nucleic acid regions in which expression of one or more genes, gene families, desired subsets of genes or alleles of genes is controlled, a representative sample of sequences is obtained for each target gene be able to. The sequences can be compared to find similar and dissimilar regions. This analysis can determine regions of identity between all family members and within a subset of family members (ie, groups within a gene family). In addition, this analysis can determine regions with identity between alleles of each family member. All families, subsets of family members, individual family members, alleles of individual family members, by considering regions of identity between family member alleles, between subsets of family members, and throughout the family Target regions that identify individual alleles of subsets or family members of can be identified.

  Controlling expression can reduce the expression of one or more target genes. Reduced expression results in post-transcriptional down-regulation of the target gene, and ultimately the protein of the end product of the target gene. For downregulation, a target nucleic acid sequence is identified in which binding of RNAi to the sequence reduces target gene expression. Reduced expression of a gene refers to the absence of observable or detectable reduction in the level of protein and / or mRNA product from the target gene compared to the absence of RNAi introduction. Complete suppression / inhibition as well as partially suppressed expression of the target gene is possible with the method of the invention. “Partially suppressed expression” means that the target gene is suppressed from about 10% to about 99% (ie, the expression of the target gene is reduced), 100% is complete suppression of the target gene / Inhibition. For example, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% of the gene expression of one or more genes. % Or about 99% can be suppressed. Alternatively, expression is suppressed or inhibited below detectable limits.

  In other embodiments of the invention, controlling expression can increase the expression of one or more genes. Increased expression can occur as detailed in the examples below. In this aspect of the invention, the target nucleic acid and the gene of interest may be separate sequences. In mRNA products from one or more target genes compared to that without protein level or RNAi introduction, increased expression of the gene is one or more target genes compared to those without RNAi introduction. Refers to the presence or observable increase in the level of protein and / or mRNA product from Increased expression of the gene means that the measurable amount of the expressed target gene is increased by any amount compared to the absence of RNAi introduction. For example, the level of gene expression is 2 fold, about 5 fold, about 10 fold, about 50 fold, about 100 fold, about 500 fold, about 1000 fold or about 2000 fold higher than occurs in the absence of interfering RNA. Can be doubled.

  In yet another aspect of the invention, by controlling expression, when one or more genes are placed under environmental conditions that generally result in reduced or increased expression of the one or more genes, The expression of one or more genes can be maintained. The expression of one or more genes can be maintained under environmental conditions that usually increase or decrease gene expression, compared to expression prior to the presence of environmental conditions that would otherwise increase or decrease expression. Resulting in a steady level of gene expression (ie, no increase or decrease in expression over time). Examples of environmental conditions that can increase gene expression include, but are not limited to, the presence of growth factors, increased glucose production, high fever and cell cycle changes. Examples of environmental conditions that can reduce gene expression include, but are not limited to, hypoxia, hypothermia, lack of growth factors, and glucose depletion.

  Quantification of gene expression allows determination of the degree of inhibition (or enhancement) of gene expression in cells or animals that contain one or more RNAi molecules. Lower doses of injected material and longer periods after administration or incorporation of RNAi may result in inhibition or enhancement in a smaller percentage of cells or animals (eg, at least 10%, 20% of target cells or animals) , 50%, 75%, 90% or 95%). Quantification of gene expression in cells or animals may show a similar amount of inhibition or enhancement at the level of target mRNA accumulation or target protein translation. The efficiency of inhibition or enhancement can be determined by assessing the amount of gene product in a cell or animal using any method known in the art. For example, mRNA can be detected with a hybridization probe having a nucleotide sequence outside the region used for the interfering RNA, or the translated polypeptide can be detected with an antibody raised against the polypeptide sequence in that region. Can do. Methods for quantifying mRNA and polypeptide are well known in the art. See, for example, Sambrook, J. et al. "Molecular Cloning: A Laboratory Manual," 2nd addition, Cold Spring Harbor Laboratory Press, Plainview, N.Y. (1989).

  The invention also relates to the control of expression of a family of genes. The term “family of genes” refers to one or more genes having similar functions, sequences or phenotypes. A family of genes may contain conserved sequences, ie, nucleotide sequences that are the same or highly homologous among all members of the gene family. In certain embodiments, an RNAi sequence can hybridize to this conserved region of a gene family, and thus one RNAi sequence can target two or more members of a gene family.

  The methods of the invention can also be used to control the expression of genes within an evolutionarily related family of genes. An evolutionarily related gene is a gene that is separated from a common ancestral gene sequence and may or may not itself be a sequence encoding one or more mRNAs. Within this evolutionarily related family, there can be a subset of genes, and within this subset, there can be conserved nucleotide sequences. The present invention also provides a method for controlling the expression of a subset of this gene by targeting RNAi molecules to this conserved nucleotide sequence. Evolutionarily related genes that can be controlled by the methods of the present invention may be endogenous or exogenous in a cell or animal and may be a member of a family of viral genes. Also, a family of viral genes that can be controlled by the methods of the invention can have family members that are endogenous in the cell or animal.

  In other embodiments, the methods of the invention can be used to control the expression of a gene or family of genes that is endogenous in a cell or animal. An endogenous gene is any gene that is inherited as an integral element of the genome of an animal species. Control of endogenous genes by the methods of the present invention can provide a way to suppress or enhance the phenotype or biological state of a cell or animal. Endogenous genes that can be controlled by the method of the present invention are not limited, but include endogenous genes necessary for cell survival, endogenous genes necessary for cell replication, endogenous genes necessary for virus replication, immunity An endogenous gene encoding a globulin locus and an endogenous gene encoding a cell surface protein. Further examples of endogenous genes include developmental genes (eg, adhesion molecules, cyclin kinase inhibitors, Writ family members, Pax family members, winged helix family members, Hox family members, cytokines / lymphokines and their receptors, growth / differentiation factors and Its receptors, neurotransmitters and their receptors), tumor suppressor genes (eg, APC, BRCA1, BRCA2, MADH4, MCC, NF1, NF2, RB1, TP53 and WTI), and enzymes (eg, ACC synthase and oxidase, no ACP) Saturating enzyme and hydroxylase, ADP glucose pyrophosphorylase, ATPase, alcohol dehydrogenase, amylase, amyloglucosidase, cataler Cellulase, chalcone synthase, chitinase, cyclooxygenase, decarboxylase, dextrinase, DNA and RNA polymerase, galactosidase, glucanase, glucose oxidase, granule-bound starch synthase, GTPase, helicase, hemicellulase, integrase, inulinase, invertase, Isomerase, kinase, lactase, lipase, lipoxygenase, lysozyme, nopaline synthase, octopine synthase, pectinesterase, peroxidase, phosphatase, phospholipase, phosphorylase, phytase, plant growth regulator synthase, polygalacturonase, proteinase and peptidase, planase (Pullanase), recombinase, reverse transcriptase, R BISCO, including a topoisomerase and xylanase).

  In other embodiments, it may be desirable to control (modulate) the tumor antigens of the cells so that, for example, the host immune system can detect these tumor cells. Many tumor antigens are well known in the art. For example, Van den Eynde BJ, van der Bruggen P. Curr Opin Immunol 1997; 9: 684-93, Houghton AN, Gold JS, Blachere NE. Curr Opin Immunol 2001; 13: 134-140, incorporated herein by reference. , Van der Bruggen P, Zhang Y, Chaux P, Stroobant V, Panichelli C, Schultz ES, Chapiro J, Van den Eynde BJ, Brasseur F, Boon T. Immunol Rev 2002; 188: 51-64. Many antibodies directed against tumor antigens are also commercially available.

  Non-limiting examples of tumor antigens include tumor antigens caused by mutations such as: alpha-actinin-4 (lung cancer), BCR-ABL fusion protein (b3a2) (chronic myeloid leukemia), CASP-8 (head and neck squamous cell carcinoma) ), Beta-catenin (melanoma), Cdc27 (melanoma), CDK4 (melanoma), dec-can fusion protein (myeloid leukemia), elongation factor 2 (pulmonary squamous cell carcinoma), ETV6-AML1 fusion protein (acute lymphoblasts) Leukemia), LDLR-fucosyltransferase AS fusion protein (melanoma), overexpression of HLA-A2d (renal cell carcinoma), hsp70-2 (renal cell carcinoma), KIAAO205 (bladder tumor), MART2 (melanoma), MUM-1f (Melanoma), MUM-2 (melanoma), MUM-3 (melanoma) Ma), neo-PAP (melanoma), myosin class I (melanoma), OS-9g (melanoma), pml-RARalpha fusion protein (promyelocytic leukemia), PTPRK (melanoma), K-ras (pancreatic adenocarcinoma) ), N-ras (melanoma) and the like. Examples of differentiated tumor antigens are not limited: CEA (gastrointestinal cancer), gp100 / Pmel17 (melanoma), kallikrein 4 (prostate), mammaglobin-A (breast cancer), Melan-A / MART-1 (melanoma) , PSA (prostate cancer), TRP-1 / gp75 (melanoma), TRP-2 (melanoma), tyrosinase (melanoma). Overexpressed or underexpressed tumor antigens include, but are not limited to: CPSF (ubiquitous), EphA3, G250 / MN / CAIX (stomach, liver, pancreas), HER-2 / neu, intestinal carboxylesterase (liver, intestine, kidney) ), Alpha-phytoprotein (liver), M-CSF (liver, kidney), MUC1 (glandular epithelium), p53 (ubiquitous), PRAME (testis, ovary, endometrium, adrenal gland), PSMA (prostate, CNS, Liver), RAGE-1 (retina), RU2AS (testis, kidney, bladder), survivin (ubiquitous), telomerase (testis, thymus, bone marrow, lymph node), WT1 (testis, ovary, bone marrow, spleen) CA125 (ovary).

  The methods of the invention can also be used to control the expression of specific alleles. Alleles are polymorphic variants of genes that occupy the same chromosomal locus. The methods of the invention allow control of one or more specific alleles of a gene or family of genes. In this aspect, the RNAi sequence may be prepared such that one or more specific alleles of a gene or family of genes are controlled, but not other additional alleles of the same gene or family of genes. it can.

It is the schematic which showed the basics of antisense transcription | transfer. FIG. 2 is a schematic diagram showing two possibilities for altering sense transcript levels by causing antisense knockdown. FIG. 3A is a schematic diagram of thymidylate synthase (TS-sense) and rTSα antisense mRNA, and FIG. 3B shows the structure of the vector used for transfection and S-AS RNA production. It is the fluorescence image which showed single cell RNA expression of TS transcript. FIG. 6 is a graph showing endogenous single cell mRNA expression of TS sense and antisense molecules. A graph showing the cellular localization of TS sense (TS) and its antisense (rtSα), HIF sense (HIF) and its antisense (aHIF) in three cell lines (HeLa, SK-N-MC and HEPG2) is there. FIG. 5 is a blot showing RPA of cytoplasmic RNA. A Northern blot for the Dicer product is shown. It is the graph which showed the expression of IFN (beta) and OAS2 mRNA. 1 is a graph showing expression profiles of BACE-1 and BACE-1-AS in various regions of mouse brain and liver. It is the scan image of the western blot of BACE-1 protein after the process of SH-SY5Y cell by siRNA. It is the schematic which shows the genome structure of the human BACE-1 locus in the human chromosome 11q23.3 locus. FIG. 5 is a graph showing silencing of BACE-1 and BACE-1-AS transcripts by synthetic siRNA. FIG. 6 is a graph showing in vivo injection of unmodified synthetic siRNA into mouse brain. FIG. 6 shows stress-induced rearrangement of antisense transcripts. FIG. 2 is a graph showing the involvement of cyclophilin-60 in the localization / regulation of BACE-1-AS and the stability of BACE-1 and BACE-1-AS transcripts. FIG. 2 is a schematic diagram of BACE-1-AS mediating the regulation of BACE-1 and its proposed mechanism of involvement in the pathophysiology of Alzheimer's disease.

The following examples are provided by way of illustration, not limitation. Although specific examples are provided, the above description is illustrative and not restrictive. Any one or more of the features of the aspects described above can be combined in any manner with one or more features of any other aspect of the invention. Furthermore, many variations of the invention will become apparent to those skilled in the art upon review of this specification.
All publications and patent documents cited in this application are incorporated by reference in the relevant part, as if each individual publication and patent document were individually displayed for all purposes to the same extent. It is. Applicant does not admit that any particular reference is "prior art" to that invention by citing various references herein.

Examples Materials and Methods
In situ hybridization HeLa cells were grown overnight on the surface of silane-coated slides and fixed with 4% paraformaldehyde (pH 7.4) for 4 minutes. After the slide was air-dried, a chamber was used to facilitate treatment of adherent cells with DNase at 37 ° C. for 16 hours. DNase Master Mix contained 10 × TurboDNase Buffer (Ambion), 100 units of DNase1, 100 units of TurboDNase and 100 units of Suprasin in a final volume of 200 μl. The cells were then washed with 1 × PBS and then incubated at 95 ° C. for 5 minutes. The first strand cDNA consists of 10 × RT Buffer (Applied Biosystems), 2.5 mM MgCl ₂ , 10 mM dNTP mixture, 10 pM Random Hexamers, 100 units RNase Inhibitor and 500 units reverse transcriptase in a final volume of 200 μl. Synthesized with RT-Master Mix. The RT reaction was accomplished using the following conditions: 30 minutes at room temperature, 3 hours at 42 ° C., and 5 minutes at 95 ° C. For in situ hybridization, cells were incubated at 65 ° C. for 1 hour with a final volume of blocking buffer (10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl ₂ , 1% Triton-X, 20 μM Random DNA). In 200 μl). After blocking, the cells were hybridized with specific probes (sequences shown in Tables 1 and 3) that span 10 μM introns at 70 ° C. for 1 hour. The slide was then washed twice with pre-warmed PBS.

Dilutional single cell RT-PCR
HeLa cultures were diluted into several cells in each bright field. RNA was extracted from 15 individual cells collected under the guidance of a confocal microscope. First strand cDNA synthesis was performed from RNA using SMART and CDSIII 3 ′ oligonucleotides from Clontech and Powerscript reverse transcriptase according to the manufacturer's instructions. This first strand cDNA was then used for PCR amplification using LD primer, DSIII PCR primer and Advantage2 Polymerase mix from Clontech cDNA library kit.

Cell extract preparation and fractionation Cytoplasmic extracts were prepared from HeLa cells transfected with various vectors. Cells were harvested after 24 hours of transfection and centrifuged at 1000 g for 5 minutes at 4 ° C. The cell pellet was washed three times with ice-cold PBS, pH 7.2, and lysis buffer (20 mM Tris-HCl, pH 7.4, 200 mM NaCl, 14 mM MgCl ₂ , 20 units Suprasin, 3 times the cell volume). , 100 units protease inhibitor, 100 μg / ml cyclohexamide, 0.1% (v / v) Triton X-100) for 10 minutes on ice. Nuclei were isolated by centrifugation at 5000g for 10 minutes at 4 ° C. The supernatant contained a cytoplasmic extract, which was immediately used for RNA extraction with Trizol (Invitrogen). Nuclear extracts were prepared by washing the pellet once with cell lysis buffer and twice with 1 × PBS, pH 7.2. Nuclear RNA was then recovered using Trizol reagent. Purity (> 98%) and nuclear integrity were determined with a microscope.

Ribonuclease protection assay (RPA)
Using the Direct Protect Lysate RPA kit from Ambion, cytosolic lysate was treated with RNase cocktail buffer and incubated with RNase A and T cocktail for 30 minutes at 37 ° C. Nucleases were removed by incubation with sacrosyl sodium and proteinase for 30 minutes at 37 ° C. RNA was precipitated with 99% ethanol and glycogen blue, then DNase treated with TurboDNase (Ambion) and then separated with 10% denatured PAGE / 8M urea.

Northern blot for Dicer product Total RNA was recovered using Trizol (Invitrogen) and precipitated with 99% ethanol. 30 μg of total RNA was loaded per lane and separated on a 10% PAGE / urea gel. The RNA was then transferred to a nylon membrane (Amersham) and blocked with salmon sperm DNA for 6 hours. The blocked membrane was hybridized overnight with a radiolabeled S-AS probe that spans the overlapping region of the TS and rTSα genes. Probes were generated by random priming of overlapping DNA using ³² P-labeled nucleotides and Amersham random priming kit. All membranes were washed once with low stringency buffer and twice with high stringency buffer for 1 hour each and the signal was detected with a Typhoon fluorescence imager.

Cell culture and transfection HeLa cells were cultured in D-MEM supplemented with 10% FBS. Exponentially growing cells were transfected with a plasmid containing the luciferase gene with either the sense or antisense overlap region or both. Cells were used for further experiments 24 hours after transfection. The pGL3 control vector (Promega) was used for the production of all S-AS constructs. We have established Pst1 and EcoR1 restriction sites downstream of firefly luciferase for cloning. Hairpins were formed between the overlapping regions using the BamH1 sequence to construct a vector with a continuous S-AS sequence (primer and probe sequences are listed in Table 3). The same vector was used as an IVT template for S-AS overlap mRNA using the MEGAscript transcription kit (Ambion).

Real-time PCR
Real-time PCR (RT-PCR) was performed with a GeneAmp 7000 apparatus (Applied Biosystems). The PCR reaction contained 20 ng cDNA, Sybrgreen or Universal Mastermix (Applied Biosystems), 300 nM forward and reverse primers, and 200 nM probe in a final volume of 50 μl (primer and probe sequences are listed in Tables 1 and 3). ). Primers and probes were designed using PrimerExpress software (Applied Biosystems). These are strand specific for each S-AS pair, and the probe covers exon boundaries to eliminate the possibility of genomic DNA amplification. The PCR conditions for all genes were as follows: 50 ° C for 2 minutes and 95 ° C for 10 minutes, 95 seconds for 15 seconds and 60 ° C for 1 minute for 40 cycles. The result is based on the cycle threshold (Ct value). The difference in Ct values between the test gene and the reference gene (either β2M or GAPDH) was calculated as ΔΔCt.

Example 1: Antisense transcript knockdown At present, the only technique that affects antisense transcript levels is through the use of siRNAs aimed at achieving effective transcript knockdown. It was. The latter term-knockdown-was introduced in the early 1990s when antisense oligonucleotides were studied (Wahlestedt, C. (1994) Antisense oligonucleotide strategies in neuropharmacology. Trends Pharmacol Sci 15 ( 2): 42-46), which is equally applicable to siRNA.

  Expression profiling revealed that sense / antisense pairs are frequently and consistently controlled. Using siRNA, we provided experimental evidence that perturbation of antisense RNA by siRNA can alter the expression of the corresponding sense messenger RNA. However, this control can be inconsistent (antisense knockdown results in increased sense transcripts) or consistent (antisense knockdown results in simultaneous decreased sense transcripts). Table 2 shows a series of human and mouse antisense transcripts targeted by siRNA. In each case, two or more siRNAs were targeted to the non-overlapping portion of the antisense strand and knockdown was confirmed using RT-PCR. Table 2 illustrates that coding antisense as well as non-coding antisense can be targeted in the same way, and that the corresponding sense transcripts in either category can be controlled in a consistent or inconsistent manner. Here we propose two novel pharmacological strategies based on knockdown of antisense RNA transcripts by siRNA (or other RNA targeting principles):

  Strategy 1: In the case of discordant control, knocking down only the antisense transcript increases the expression of the traditional (sense) gene. If the latter gene encodes a known or putative drug target, knockdown of its antisense counterpart would be able to mimic the action of a receptor agonist or enzyme stimulant. Table 2 provides examples where knockdown of antisense (coding as well as non-coding) transcripts was shown to control sense expression inconsistently. For example, in Parkinson's disease, enhanced activity of a kinase localized in mitochondria, PINK1, (eg, increased PINK1) may be desirable, and knockdown of its non-coding antisense partner may be a means for that. Furthermore, to stimulate angiogenesis, enhanced signaling through the G protein-coupled receptor (GPCR), CD97, can be achieved by targeting its (coded) antisense partner, Ddx-39.

Example of strategy 1 (increased PINK1, eg Parkinson's disease):

  Strategy 2: In the case of coordinated control, synergistic reduction of conventional (sense) gene expression is achieved by simultaneous knockdown of antisense and sense transcripts. These concepts are illustrated in FIGS. 2A and 2B. When siRNA is used to achieve knockdown, this strategy overlaps one siRNA targeted to the sense transcript and another siRNA targeted to the corresponding antisense transcript, or simultaneously. It is further tested by applying a single energetically symmetrical siRNA that simultaneously targets the sense and antisense transcripts. According to Table 2, pursuing such dual co-targeting would be appropriate in the case of hypoxia-inducible factor 1 alpha, for example, a target whose inhibition may be beneficial in various disease states. Another example in Table 2 is the adrenomedullin AM1 receptor, a GPCR that has been shown that reduced signaling is also therapeutically beneficial.

With the advent of a new functional RNA world, there are new potential drug targets to consider. Among these are a number of naturally occurring antisense transcripts that have the ability to control the expression of sense transcripts, including those encoding conventional drug targets. Since many of these antisense transcripts are non-coding RNAs, they cannot be manipulated at the protein level. By using siRNA we have shown that antisense transcript knockdown can result in either increased (consistent control) or decreased (inconsistent control) sense transcript expression. These findings and concepts form the basis for new pharmacological strategies.

Example of strategy II (simultaneous knockdown of antisense and sense transcripts for Alzheimer's disease):

Example 2: Natural antisense-mediated regulation of gene expression in mammals Naturally occurring antisense transcripts (NATs) have been reported for 20% of the human genome. Recent reports indicate that NAT is present for at least 72% of mouse transcripts. Most natural antisense transcripts are cis-coded antisense. By definition, cis-NAT is a complementary mRNA with overlapping transcription units at the same chromosomal locus. Trans NAT is complementary RNA transcribed from different chromosomal loci. A chimeric transcript is an mRNA having identity to two or more regions of the genome, and may be an artifact of cDNA library construction. More than 70% cis-NAT is a tail-to-tail type with a 3 ′ overlap, while 15% is a head-to-head type with a 5 ′ overlap region. The remaining molecules have introns or coding sequence overlap. Many NATs do not display an open reading frame and are classified as non-coding RNA.

  Interactions with antisense and corresponding sense transcript partners do not follow a uniform and predictable pattern. The interaction between two NATs targeting the human genes, HIF-1α and TS, was investigated. Antisense transcripts of HIF (aHIF) are non-coding RNAs that can change the splicing of HIF and also the ratio between the two splicing forms of HIF. Specifically, it was postulated that antisense molecules could destabilize one splice variant of HIF mRNA and shift the balance to the other variant side. Editing is another proposed function of NAT through the conversion of adenosine to inosine nucleotides in the pre-mRNA. The antisense sequence of TS (rTSα) induces editing of the sense RNA molecule, thereby driving downregulation of TS mRNA. Importantly, the NAT of TS encodes a protein, but there is no open reading frame expected for aHIF. These two known candidates from the coding and non-coding subgroups of NAT that could potentially regulate sense mRNA through two different modes of action were selected for the study.

  One of the most exciting findings in genomic biology in recent years is the discovery of RNA interference (RNAi), which has been proposed as a possible mechanism by which NAT can regulate gene expression. RNAi is an intrinsic cellular process that is activated when double-stranded RNA (dsRNA) enters the cell. RNAi, originally discovered in Caenorhabditis elegans, is an evolutionarily conserved, post-transcriptional gene silencing mechanism. dsRNA is processed into small duplex RNA molecules of about 21-22 nucleotides called small interfering RNA (siRNA) by an RNase III enzyme called Dicer. The siRNA molecule then interacts with a multiprotein complex called the RNA-induced silencing complex (RISC), resulting in a sequence-specific association of the activated RISC and related RNA transcripts. This interaction leads to sequence-specific cleavage of the target transcript. It has been suggested that dsRNAs derived from endogenous S-AS duplexes can act as Dicer substrates and then act via the RNAi pathway by generating siRNAs. The siRNA then controls one or both of the S-AS transcripts.

  In summary, NAT has been proposed to control gene transcription, RNA splicing, polyadenylation, editing, stability, transport and translation. The purpose of this study was to investigate the mechanism of NAT action. The common complementary region in the exon of NAT implies the possibility of duplex formation in the cytoplasm, and the intron overlap sequence suggests a nuclear dsRNA duplex. Theoretically, all proposed regulatory mechanisms require RNA duplex formation in the cytoplasm or nucleus, so there is no cellular evidence for RNA duplexes using HIF and TS as model genes. It was the main focus of this study.

Results: An in situ hybridization method was used to assess the simultaneous presence of both endogenous TS and rTSα. HeLa cells were grown on the slide surface, fixed and treated with DNase (see Materials and Methods). First strand cDNA was synthesized and subjected to in situ hybridization using a probe that spans the strand-specific intron. (A schematic of the TS sense-antisense gene and probe is shown in FIG. 3A). Importantly, detection of contaminating DNA is eliminated by using a probe that spans introns, the probe covers at least part of the overlap region of both transcripts, and the signal is derived from full-length mRNA. It is guaranteed. The results show that both transcripts co-exist in a single cell (Figure 4).

In order to demonstrate the coexistence of S-AS pairs in single cells as a concept that conflicts with cell populations, a method was designed to detect co-expression of NAT in single cells. RNA was extracted from single cells under microscopic guidance and TS and rTSα transcripts were quantified by real-time PCR using TaqMan technology (FIG. 5). Primers were strand specific for both sense and antisense. S-AS expression was normalized to beta2-microglobulin (β ₂ M), an extremely abundant mRNA, as an internal control. The sensitivity of the method was assessed by comparing the expression of TS and rTSα with that of a relatively abundant gene, TATA binding protein (TBP). As shown in FIG. 5, TS and rTSα were 7% of β ₂ M expected for genes with low expression, and the level of TBP was 5% relative to β ₂ M. Thus, all of the S-AS transcripts were present at approximately similar levels in single cells.

  Next, the intracellular location of TS and HIF transcripts was investigated. Cytoplasmic and nuclear extracts were prepared from HeLa cells and used immediately for RNA extraction. RNA was then reverse transcribed and used for quantification of S-AS transcripts by real-time PCR. Importantly, the sense strands of both genes had similar expression levels in the cytoplasm and nucleus, in contrast, antisense transcript levels were compared to those detected in the cytoplasm in the nucleus. It was 1000 times higher. Thus, these data suggest a spatial divergence of S-AS pairs (FIG. 6).

  Next, S-AS duplex formation in the cytoplasm of HeLa cells was investigated using a ribonuclease protection assay (RPA). Although HeLa cells endogenously express both S-AS mRNA, three vectors were constructed that generate sense, antisense or continuous S-AS overlap mRNA in eukaryotic cells (FIG. 3B). For two of the constructs, the 3 'overlap region of TS and rTSα was placed downstream of the luciferase gene, thereby allowing transfection efficiency to be monitored. These two vectors were cotransfected into HeLa cells to generate a state in which RNA with overlapping regions was overexpressed. For the third construct, the sense and antisense complement regions were designed in the same vector with a short hairpin between the S-AS overlaps. RNA from this vector is thought to fold relative to itself in the cell to form an RNA duplex. As an additional control, in vitro transcription (IVT) of the vector was performed to create an artificial RNA duplex and then transfected into cells. To investigate the presence of RNA duplexes in transfected and untreated cells, cytoplasmic lysates were isolated, then treated with RNAse A and T, and then separated on a polyacrylamide gel. Existing RNA duplexes were detected with a radiolabeled probe against the S-AS overlap region. S-AS duplex was detected in cells transfected with IVT dsRNA. No RNA duplexes were detected in cells overexpressing S-AS or expressing endogenous levels of NAT (FIG. 7). These data suggest that endogenous NAT as well as synthetically overexpressed S-AS RNA did not form duplexes in the cytoplasm of HeLa cells. It is possible that the expected RNA duplex in living cells is transient and unstable and is rapidly processed into endogenous siRNA or other intermediate products. To investigate this possibility, Northern blot analysis with a radiolabeled probe across the overlapping region of S-AS mRNA was designed. These randomly designed probes that can have the potential to detect S-AS sequences of any length, from full-length RNA to less than 20 bp Dicer products, are used to probe for the presence of processed RNA. It was. The hypothesis was that if RNA duplexes were present, they would eventually be processed by Dicer into 21 base pair RNA oligonucleotides. HeLa cells were transfected with the same vectors used in the previous experiments producing sense, antisense or S-AS RNA. RNA duplexes from the S-AS overlap region produced by IVT were used as a positive control and were transfected into cells. The Dicer product was only present in cells transfected with IVT dsRNA or cells transfected with a vector that produced an internal hairpin dsRNA (FIG. 8). A positive band was detected at 1100 bp (full length RNA generated from the vector) in overexpressed cells and 200 bp in IVT RNA transfected cells. However, the lack of a 21 bp RNA molecule in untransfected or overexpressing cells suggests that the S-AS duplex was not processed by Dicer.

  The interferon signaling cascade is part of the cellular antiviral defense mechanism and can be triggered by dsRNA. Interferon β (IFNβ) and 2 ′, 5′-oligoadenylate synthetase-2 (OAS2) mRNA levels were measured in cells overexpressing the S-AS transcript (FIG. 9). IFNβ mRNA levels were upregulated 10,000-fold in cells transfected with in vitro transcribed dsRNA, but were unchanged in cells with overexpressed S-AS transcripts. OAS2 levels were also upregulated approximately 600-fold only in cells transfected with IVT double-stranded RNA. Although these data indicate that no cytoplasmic RNA duplex is formed by S-AS mRNA, it is possible that the interferon pathway may be unresponsive to the intracellular RNA duplex.

  Taken together, this study suggests that NAT does not form cytoplasmic RNA duplexes that operate the RNAi mechanism. Overlapping transcripts in the antisense direction, encoding or not encoding proteins, have the potential to form dsRNA, a substrate for multiple different RNA regulatory pathways. One prominent pathway for dsRNA is its degradation into small RNAs by the Dicer enzyme complex. Several experimental approaches have been used in an attempt to identify the presence of RNA duplexes in the cytoplasm of cells and detect Dicer products involved in dsRNA processing. Results with synthetic S-AS constructs and endogenous NAT did not support the presence of cytoplasmic RNA duplexes or involvement of RNAi mechanisms.

  The simultaneous presence of both S-AS mRNAs is a requirement for NAT control, and many of the NAT candidates predicted in silico can be excluded by this criterion alone. The expression level of S-AS is important because these allow prediction of the mode of control. The high level of S-AS in single cells suggested by our experimental model is contrary to the involvement of RNAi. However, another explanation of this phenomenon is RNA-mediated control of other types of gene expression without blocking translation or changing mRNA levels. Expression assessment and mRNA level assessment are recommended as a first step in the study of other prospective S-AS candidates.

  While changes in antisense transcript levels can affect sense mRNA levels, changes in S-AS are not necessarily reciprocal. Recently, we showed that knockdown of antisense transcripts increased sense transcript levels, but no reverse interaction was observed. This finding suggests that antisense mRNA is involved in the regulation of sense transcripts, but sense mRNA does not appear to regulate antisense expression. If endogenous RNAi is involved in mammalian S-AS events, it can be expected that both transcripts will show similar expression profiles in knockdown experiments.

  In general, the above findings are consistent with the conclusion that S-AS gene regulation is not involved in the RNAi mechanism. In fact, further support comes from two other observations. First, small RNA molecules were not detected even for highly expressed S-AS, suggesting dicer-independent RNA processing, and second, the interferon cascade was not activated by NAT. Indeed, if at least 70% of mammalian genes have NAT and the mechanism is through RNA duplex formation, it can be expected that there will be a cumulative interferon response. Our study showed dramatic induction of β-interferon and OAS2 mRNA by dsRNA transfection, but this was not seen in cells overexpressing S-AS, indicating the lack of NAT duplexes. It is shown.

  To date, there is no report on endogenous mammalian siRNA derived from NAT (Makalowska I, Lin CF, Makalowski W: Overlapping genes in vertebrate genomes. Comput Biol Chem 2005, 29 (1): 1-12). However, endogenous siRNA can be programmed into the RISC, the effect of which is long-lasting, and can lead to down regulation of the target RNA. Theoretically, a 500 bp dsRNA produces a library of siRNAs. This collection of siRNAs can impair protein production at two levels, either by degrading many “off targeted” mRNAs or by blocking translation. This range of non-specific effects is much greater considering the large number of genes known to have antisense sequences.

  Consistent with the data in this study, the presence of endogenous miRNA has been reported, but no endogenous mammalian siRNA has been described so far. This finding is also contrary to the processing of the endogenous RNA duplex in the dicer-dependent pathway, further demonstrating our findings.

Our data suggest that antisense expression is not linked to the transcript degradation pathway. However, our method completely excludes any function proposed for the formation of RNA duplexes in the cell nucleus or the regulation of gene expression by NAT, such as editing, nuclear retention, splicing or transport. Not done. Many different functions and mechanisms have been suggested for NAT, but no systematic approach has been made to date for the taxonomy or prediction of the mechanisms suggested. Our research can be the start of a functional approach to NAT research that can lead to the classification of NAT based on its unique bioinformatic features. Our methodology can also be extended to provide a systematic approach to natural antisense-mediated control of gene expression.

Example 2: Nuclear-retained RNA transcripts utilize β-secretase expression by a feedforward mechanism Rapid cDNA amplification (RACE): RLM-RACE ready cDNA (Ambion, Austin, TX) CDNA from human and mouse brain was used for nested PCR reactions with gene specific primers and kit primers. Mouse and human 3 ′ and 5 ′ PCR products were excised from the gel, purified and cloned into T-Easy vector (Promega). Twenty positive colonies from each series were sequenced.

Real-time PCR: Real-time PCR (RT-PCR) was performed with a GeneAmp 7900 apparatus (Applied Biosystems). The PCR reaction contained 20-40 ng cDNA, Universal Mastermix (Applied Biosystems), 300 nM forward and reverse primers, and 200 nM probe in a final volume of 15 μl (primer and probe sequences are listed in Tables 1 and 3). ). Primers and probes were designed using FileBuilder software (Applied Biosystems). These are strand specific for the S-AS pair, and the sense probe covers exon boundaries to eliminate the possibility of genomic DNA amplification. PCR conditions for all genes were as follows: 50 ° C for 2 minutes and 95 ° C for 10 minutes, then 40 cycles of 95 ° C for 15 seconds and 60 ° C for 1 minute. The result is based on the cycle threshold (Ct value). The difference in Ct values between the test gene and the reference gene (18s rRNA) was calculated as ΔΔCt.

Cell culture and transfection: SH-SY5Y cells are a mixture of MEM and F12 supplemented with 10% FBS, 1% NEAA, 1% L-glutamate and 1% sodium bicarbonate (medium). Cultured in (culture medium). Cells in logarithmic growth phase were transfected with 20 nM siRNA using 0.2% Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen). The cells were incubated for 48 hours before further use. Cells were exposed to 20 μM retinoic acid (Sigma) for 2 weeks for induction of neuron-like differentiation. For stress induction, cells are suspended for 5 minutes in medium containing 30 mM KCl or 2 hours in medium containing Aβ1-42 peptide (Tocris Co. California, USA) and then for fractionation as described below. Processed. For rescue experiments, cells were washed with PBS and resuspended in culture medium for 1 hour at 37 ° C. Control samples were treated similarly and used for parallel cell fractionation and RNA extraction. Parental CHO and CHO-7PA2 cells were grown in DMEM containing 10% FBS. For habituation, CHO-7PA2 cells were maintained in serum-free medium and supernatant medium was collected after 24 hours. SH-SY5Y cells were exposed to conditioned media 24 hours prior to fractionation. Control cells were treated with similar conditioned media from parental CHO cells.

Northern blot: Total RNA was isolated using Trizol reagent and 10 μg total RNA per lane was loaded onto a 1% agarose gel. The RNA was then transferred to a Hybond membrane (Ambion) and blocked with Ultrahybrid (Ambion) for 6 hours. The blocked membrane was hybridized overnight with a radiolabeled S-AS probe that spans the overlapping region of mouse BACE-1 and BACE-1-AS. Probes were made by random priming of overlapping DNA using 32P labeled dCTP nucleotides and Amersham random priming kit. The membrane was washed once with a low stringency buffer and twice with a high stringency buffer, each for 10 minutes, and the signal was detected with a fluorescence imager.

Cell extract preparation and fractionation: Cytoplasmic extracts were prepared from SH-SY5Y cells. Cells were harvested and centrifuged at 1000 g for 5 minutes at 4 ° C. The cell pellet was washed once with ice-cold PBS, pH 7.2, and lysis buffer (20 mM Tris-HCl, pH 7.4, 200 mM NaCl, 14 mM MgCl ₂ , 3 times the cell volume) on ice for 10 minutes. Dissolved in 20 units Suprasin and 0.1% (v / v) Triton X-100). Nuclei were isolated by centrifugation at 500 g for 10 minutes at 4 ° C. The supernatant contained a cytoplasmic extract that was immediately used for RNA extraction. Nuclear extracts were prepared by washing the pellet once with cell lysis buffer. Nuclear RNA was then recovered using the RNAeasy mini kit.

RNA pull-down and mass spectrometry: Hypotonic fractionation buffer with high MgCl ₂ concentration was used for homogenization with Dounce type homogenizer for cell disruption. The SH-SY5Y cell lysate was immediately hybridized with strand-specific biotin-labeled RNA probes for BACE-1 and BACE-1-AS, and then incubated with streptavidin beads for 15 minutes. The purified protein was separated by polyacrylamide gel electrophoresis (PAGE). Mass spectrometry was used to detect proteins purified with RNA transcripts. An RNA probe targeting Prltk that does not match the human genome was used as a control.

Stability and α-amanitin treatment: HEK-293T cells were plated in 24-well plates. After 24 hours, cells were treated with 5 μg / ml α-amanitin and incubated with conditioned medium from 7PA2 or CHO control cells. Cells were harvested 6, 12, and 24 hours after treatment for RNA purification and RT-PCR. Three independent samples were taken at each data point and all samples had untreated and untransfected counterparts for RNA purification and data analysis.

Statistical analysis: All experiments were performed with 6-20 biological replicates and 3-6 technical replicates. Data are shown graphically as a comparison with the control treatment group after a post-test of treatment factors using the main effect in a two-factor analysis of variance (ANOVA). Calculated as the significance p-value for each treatment and displayed in each graph, p <0.05 was considered significant.

RNA fluorescence in situ hybridization (RNA-FISH): SH-SY5Y cells were grown overnight on silane-coated slides and fixed with 4% paraformaldehyde (pH 7.4) for 4 minutes. Cells were permeabilized with 0.2% Triton-X for 4 minutes at room temperature. After air-drying the slides, easy treatment of adherent cells with prehybridization buffer (50% formamide, 5 × SCC, 50 μg / ml salmon semen DNA and 0.1% Tween-20) at 65 ° C. for 1 hour A chamber was used to achieve this. Prltk mRNA (as a negative control) and biotinylated probes for non-overlapping parts of BACE-1 and BACE-1-AS were cloned S-AS RNA using the T7 promoter and BiotinUTP with the MEGAScript kit (Ambion). Of in vitro transcription. The probe was then added to hybridization buffer (2.5 μg / ml) and incubated at 65 ° C. for 6 hours. The slides were then washed 3 times with PBS at 65 ° C. for 10 minutes each.

Western blot: HEK-SW cells were transfected with 20 nM BACE-1 sense, BACE-1-AS or siRNA of both transcripts. 48 hours after transfection, the cells were lysed with 200 μl of Laemmli sample buffer from BioRad containing 350 mM DTT. 20 μl of cell lysate was then separated on 10% SDS PAGE and transferred to a nitrocellulose membrane overnight. The membrane was incubated with a primary antibody against BACE-1 (from Abcam) and a secondary antibody conjugated to HRP. After the addition of HRP substrate, the chemiluminescent signal was detected with X-ray film. The same membrane was peeled off and reused for detection of β-actin as a loading control.

ELISA and HTRF assays: HEK-SW cells artificially overexpressed with APP Swedish mutations were seeded on 6-well plates and after 24 hours 20 nM against BACE-1 sense, BACE1-antisense or both transcripts Of siRNA. Media from cultured cells was harvested 48 hours after transfection for sandwich ELISA using Aβ1-42 antibody. Capture antibody (mouse monoclonal antibody against amyloid Aβ, Abcam) was incubated overnight at 4 ° C. in a Maxisorb 96 clear plate in the presence of carbonate buffer. The collected medium was added to the plate and incubated with primary antibody (Aβ1-42 from Abcam) and goat anti-rabbit IgG secondary antibody. After addition of HRP substrate, luminescence was detected using a SpectraMax plate reader. The average absorbance at 405 nm of 3 replicates was subtracted from the background and normalized to the negative control siRNA sample. A time-resolved fluorescence (HTRF) assay (Cisbio) was used for detection of Aβ1-42. When two specific monoclonal antibodies against Aβ1-42 are labeled with a fluorophore and simultaneously bound to an Aβ peptide, the emission of the first fluorophore excites the second fluorophore based on the distance between them. The HTRF reaction was performed in a single tube with APPtg and purified protein from wild type mice, without a washing step, according to the product protocol, thereby allowing direct measurement of peptides.

Animal experiments: After IACUC approval for animal experiments at The Scripps Research Institute, 18 6 month old male mice were used for in vivo experiments. Mice were divided into 3 groups of 6 animals, and osmotic minipumps (Alzet) were surgically implanted with a chronic indwelling cannula in the dorsal side of the third ventricle. Osmotic minipumps are BACE-1 (Group 1), BACE-1-AS (Group 2), or control siRNA (previously known to have no effect on the entire human and mouse genes, Group 3) Results in continuous infusion (0.25 μl / hr) of siRNA at a dose of 0.4 mg / day. After 14 days of continuous siRNA infusion, mice were euthanized and brains were removed. For quantitative RNA measurements, 5 tissues from each mouse were removed: dorsal hippocampus, ventral hippocampus, dorso-medial prefrontal cortex, dorsal striatum and cerebellum. Tissues were excised, rinsed with ice-cold PBS, and RNA was extracted after homogenization into Trizol reagent (Invitrogen) according to the manufacturer's protocol. The extracted RNA sample was passed through a Qiagen RNeasy column and subjected to on-column DNAse treatment to remove DNA contaminants. The concentration of RNA samples was determined spectrophotometrically and 800 ng of each sample was used for first strand cDNA synthesis using random hexamers and reverse transcriptase (Applied Biosystems) in a final volume of 40 μl. Real-time PCR (RT-PCR) measurements were performed as described above. It was done as described above. Percentile changes in RNA levels of individual tissues compared to control mice were plotted in each graph.

  In another experiment, 4 male APP-tg mice (Tg 19959) and 4 control littermates, all 6 weeks old, were sacrificed. Brain tissue was used for RNA measurement and Aβ1-42 detection by HTRF.

Identification of the BACE-1 non-coding natural antisense transcript: The genomic structure of the human BACE-1 locus is illustrated in FIG. 12A. The location of our primer, probe and siRNA sequences is shown in FIG. 12B and listed in Table 5. BACE-1-AS transcripts are highly conserved across species (see FIG. 12B).

  Sequence information regarding human BACE-1 (NM_012104), mouse BACE-1 (NM_011792), human BACE-1-AS (CB960709) and mouse BACE-1-AS (AK074428 and AK078885) can be found on the UCSC Genome Bioinformatics website (genome. ucsc.edu/cgi-bin/hgGateway). In order to fully characterize the mechanism of the BACE-1-AS genomic structure, we performed rapid amplification of cDNA ends (RACE) experiments for directional sequencing from the 5 'and 3' ends of NAT. Went. For human BACE-1-AS, RACE revealed a sequence almost identical to the expressed sequence tag (EST) from the UCSC database (CB960709), with the exception of the additional nucleotides at its boundaries shown in FIG. 12C. Two splice variants were identified for human and mouse BACE-1-AS, which overlapped with the BACE-1 sense transcript in both species. Two separate ESTs were also found for mouse BACE-1-AS, which matched a single contig sequence covering exons 5 and 6 of the mouse BACE-1 gene. (FIG. 12D). Poly A tail and cap structures have been found for both human and mouse antisense sequences, indicating that BACE-1-AS is the product of RNA polymerase II and is a fully processed RNA transcript. It is a suggestion. However, the absence of an open reading frame suggested that BACE-1-AS is an ncRNA. Also, multiple mismatches with the genomic sequence were detected in both human and mouse BACE-1-AS, as the “A to I” editing was due to intranuclear duplex formation by the sense transcript (Kawahara and Nishikura, 2006 FEBS Lett 580, 2301-2305). This latter finding may be related to the BACE-1-AS nuclear retention pattern, as will be explained in detail below. All new sequences of human and mouse BACE-1-AS are also shown in the additional data section.

Expression profiling: Using RT-PCR, we measured the relative expression of BACE-1 (sense) and BACE-1-AS (antisense) RNA transcripts in various regions of mouse brain and liver. Both transcripts were 2-5 times more abundant in various regions of the brain compared to the liver. Cerebral cortex and amygdala showed the highest expression of both transcripts in the sampled brain area (FIG. 10A). Northern blot analysis confirmed the expression of BACE-1 and BACE-1-AS in mouse brain tissue (FIGS. 10A, 10B). BACE-1 and BACE-1-AS transcripts were also expressed in undifferentiated and differentiated human neuroblastoma SH-SY5Y cells. Induction of differentiation of human neuroblastoma SH-SY5Y cells is accompanied by approximately 50% decrease in BACE-1-AS transcript expression and approximately 20% decrease in BACE-1 transcript (FIG. 10B). This suggests that the expression of both sense and NAT transcripts is consistent.

BACE-1-AS knockdown consistently reduced BACE-1 sense transcripts in vitro: We next analyzed the expression of its sense partner (BACE-1 mRNA) in BACE-1-AS. The role in regulation via siRNA knockdown was investigated. FIG. 13A shows three different siRNA sequences that knocked down the BACE-1-AS transcript by ˜60% in human SH-SY5Y cells. All three siRNA sequences target the antisense transcript (but not BACE-1 mRNA), and the sense transcript is consistent and coincident with the antisense transcript It is very effective for knocking down. Since three different siRNA molecules that target the antisense transcript (BACE-1-AS) result in consistent knockdown of BACE-1, siRNA is indirectly directed by a non-specific (or “off-target”) mechanism. In particular, it is unlikely that the BACE-1 transcript was knocked down. We therefore show that in human SH-SY5Y neuroblastoma cells, the non-coding antisense transcript has a regulatory effect on the expression of BACE-1 mRNA and that BACE- without targeting the BACE-1 transcript itself. It suggests that knocking down 1-AS can affect the expression of BACE-1.

  The effect of knocking down BACE-1 mRNA was also evaluated. FIG. 13A shows sense-targeted siRNA (Sa) in which BACE-1 mRNA is knocked down 70% in SH-SY5Y cells. Two other siRNA sequences targeting BACE-1 mRNA, Sb and Sc (shown in FIG. 12B), similarly knocked down BACE-1 mRNA. S-a targeted the non-BACE-1-AS overlapping portion of the BACE-1 sense transcript and did not alter BACE-1-AS expression 48 hours after siRNA application. Thus, in SH-SY5Y cells, BACE-1 mRNA knockdown does not regulate BACE-1-AS expression.

BACE-1-AS knockdown reduced the production of BACE-1 protein and Aβ1-42: The consistent effect on BACE-1 mRNA expression by knocking down BACE-1-AS was determined by Western blot. As determined, BACE-1 protein levels were also not restricted to mRNA levels since they were reduced by administration of NAT-specific siRNA (FIG. 11). Furthermore, we measured the level of Aβ1-42 after siRNA treatment using an ELISA method. Aβ1-42 is an enzymatic cleavage product of APP by BACE-1, and can be detected in a HEK-SW cell line containing APP having a so-called Swedish mutation. The level of Aβ1-42 was significantly reduced by treatment of the HEK-SW cell line with siRNA against BACE-1-AS (FIG. 13B).

Combined targeting of BACE-1 sense and antisense transcripts showed a synergistic effect: we then achieve synergistic effect by co-targeting BACE1 and BACE-1-AS transcripts in HEK-SW cells Investigate what can be done. Simultaneous application of siRNA to BACE-1 sense and antisense transcripts reduced Aβ peptide to a greater extent than induced by knockdown of either sense or antisense transcripts (P <0.001). ). See Figure 13B. This finding raises the possibility that some or many of the siRNA molecules are unintentionally designed to simultaneously target cissense / antisense pairs. For example, it has recently been shown that targeting BACE-1 with siRNA in vivo improves AD-like neuropathological symptoms and behavioral disorders in APP transgenic mice (Singer et al., (2005). Nat Neurosci 8, 1343-1349). The most effective siRNA molecule in this study targeted the overlapping region of the BACE-1 sense / antisense locus, which is an overall knockdown of both sense and antisense BACE-1 transcripts. It raises the possibility of contributing to effective effectiveness.

In vivo administration of siRNA targeting BACE-1 sense or antisense transcript: The above evidence demonstrates that perturbation of BACE-1-AS results in down-regulation of BACE-1 mRNA in vitro It is. Next we evaluated whether a similar relationship exists in the mouse brain in vivo. To elucidate this question, mice with a chronic indwelling cannula on the dorsal side of the third ventricle were prepared. Mice also have BACE-1 (Group 1), BACE-1-AS (Group 2), or control siRNA (previously known to have no effect on the entire human and mouse genes, Group 3) An osmotic minipump that provides continuous infusion (0.25 μl / hour) of siRNA at a dose of 0.4 mg / day for 2 weeks was subcutaneously implanted (Thakker et al., (2004). Proc Natl Acad Sci USA 101, 17270-17275, Thakker, DR, et al. (2005). Mol Psychiatry 10, 782-789, 714). The tube was connected to the outlet of the osmotic minipump so that the siRNA was delivered directly to the brain and tunneled subcutaneously to the indwelling cannula. After 14 days of continuous siRNA injection, mice were euthanized and brains were removed. The expression levels of BACE-1 and BACE-1-AS were evaluated in the dorsal hippocampus, ventral hippocampus, dorsal medial prefrontal cortex and dorsal striatum.

  Notably, BACE-1 concentrations were reduced in all four brain regions by administration of siRNA selectively directed to either BACE-1 or BACE-1-AS (FIGS. 14A-14d). SiRNA directed to either sense or antisense transcripts resulted in a simultaneous decrease in BACE-1 and BACE-1-AS levels compared to the control treatment group. Furthermore, we measured S-AS transcripts in the cerebellum as a control tissue (FIG. 14E) and found that the levels of BACE-1 and BACE-1-AS were unchanged. This is an expected result for tissues that are not in direct contact with the cerebrospinal fluid of the third ventricle. Overall, these in vivo findings replicate our in vitro findings suggesting consistent control of BACE-1 mRNA by BACE-1-AS.

BACE-1-AS transcripts accumulate in the cell nucleus: To study the intracellular distribution of BACE-1 and BACE-1-AS, we separated the lysates of SH-SY5Y cells into cytoplasm and nuclear fractions RNA was extracted for RT-PCR analysis. BACE-1-AS was abundant in the nuclear fraction compared to the cytoplasmic fraction (FIG. 15A). Intranuclear retention of BACE-1-AS was confirmed by RNA fluorescence in situ hybridization (RNA-FISH) images of SH-SY5Y cells (FIGS. 15A-15D inset). BACE-1-AS is approximately 30 times more abundant in the nucleus than in the cytoplasm, while the BACE-1 sense transcript (and the control β-actin mRNA) was almost equally distributed between the nucleus and the cytoplasm.

  We also designed RNA-FISH experiments using multicolor fluorescent probes to visualize the subcellular localization of sense and antisense transcripts. Exposure of the cells to the stressor resulted in a transition of the BACE-1-AS FISH signal (FIGS. 15A-15D inset). We utilized an RNA probe derived from Prltk luciferase that does not match the human genome as a negative control probe. As can be seen in the micrograph of FIG. 15A, the antisense transcript BACE-1-AS was mainly detectable in the nucleus. In contrast, sense BACE-1 transcripts were scattered throughout the cell.

Profiling RNA-protein interactions by mass spectrometry: In order to identify proteins that may be involved in nuclear retention and / or transport of non-coding antisense transcripts, we remove RNA and interact with RNA Profiling. The high MgCl 2 and non-denaturing properties of the method facilitate the maintenance of RNA-protein interactions during experimental manipulation. Table 4 lists proteins associated with BACE-1 and BACE-1-AS transcripts. Proteins involved in the translation process were selectively co-purified with BACE-1 mRNA. In contrast, proteins with high nuclear abundance were associated with BACE-1-AS transcripts. These findings are consistent with the non-coding nature of antisense transcripts and support its concept of nuclear retention.

Little is currently known about the mechanisms by which the expression and function of cyclophilin-60 (Cyp60) and BACE-1-AS: BACE-1 are controlled. However, recent large-scale siRNA screening studies have revealed that Cyp60 knockdown reduces BACE-1 mRNA and protein levels (Espeseth et al., (2006) Mol. Cell Neurosci. 33, 227 -235). Cyclophilin-60 (Cyp60) is a member of the cyclophilin family of peptidylprolyl isomerases. Cyclophilins form a highly conserved protein family whose members play an important role in protein folding, immunosuppression by cyclosporin A and infection of HIV-1 virions. The Cyp60 protein interacts with the proteinase inhibitor eglin c and is localized in the nucleus. Cyp60 has also been reported to be involved in cell surface expression of CD147 (also known as extracellular matrix metalloprotease inducer), suggesting that Cyp60 is involved in the migration of CD147 to the cell surface. Is done.

  In view of the proposed role of BACE-1-AS in regulating BACE-1 expression and the previously published observation that Cyp60 regulates BACE-1 mRNA expression, we also identified that Cyp60 is also a BACE-1-AS It was assumed that it might be involved in control. To test this hypothesis, we examined the impact of Cyp60 knockdown on BACE-1 and BACE-1-AS expression. First, we confirmed the role of Cyp60 in regulating BACE-1 mRNA expression by demonstrating that siRNA-mediated knockdown of Cyp60 reduced BACE-1 mRNA levels in human fetal kidney cells (HEK293T). However, BACE-1-AS transcripts were also reduced by exposure to Cyp60 siRNA (FIG. 16A). When compared to Cyp60 knockdown mediated reduction of BACE1 mRNA, the extent of BACE-1-AS transcript reduction was greater and occurred at an earlier time point (FIG. 16B). Cyp60 is almost retained only in the nucleus (Wang et al., (1996) Biochem J. 314 (Pt 1), 313-319), and BACE-1-AS is also localized in the nucleus. This suggests that Cyp60 acts selectively on BACE-1-AS and regulates the expression of BACE-1 mRNA. Consistent with this hypothesis, Cyp60 knockdown significantly altered the nuclear retention pattern of BACE-1-AS transcripts (FIG. 16C).

  The human BACE-1-AS sequence shown here is based on 5 ', 3' RACE cloning and sequencing results and is compared to the EST sequence from UCSC (cDNA CB960709).

Human BACE-1-AS major variant (10 clones) SEQ ID NO: 64:

Human BACE-1-AS short chain mutant (6 clones) SEQ ID NO: 65:

  The mouse BACE1-AS sequence shown here is based on 3'RACE cloning and sequencing results and is compared to the EST sequences from UCSC (cDNA AK077428.1 and cDNA AK078885.1).

Mouse BACE-1-AS major variant (10 clones) SEQ ID NO: 66:

Mouse BACE-1-AS long chain variant (2 clones) SEQ ID NO: 67:

Cell stress-induced changes in BACE-1-AS nuclear retention pattern: various cell stressors such as hypoxia, reoxygenation, oxidative stress and some pro-apoptotic factors have long been implicated in the pathogenesis of AD It has been said. These stressors enhance BACE-1 activity and Aβ production (Tong et al., (2005) Neural Transm 112, 455-469). The above-described nuclear retention of BACE-1-AS is reminiscent of the separation by CTN-RNA compartment recently reported by Prasanth et al. (Prasanth et al., (2005) Cell 123, 249-263). This non-coding RNA transcript, like BACE-1-AS, is mainly localized in the nucleus and contains elements for editing from adenine to inosine, an important mechanism involved in its nuclear retention. It has been shown that CTN-RNA is cleaved under cellular stress to produce the protein-encoding transcript mCAT2 mRNA. Based on these findings, we speculated that cellular stress could release BACE-1-AS from the nucleus, thereby regulating BACE-1 mRNA expression. Cell stress is considered an important trigger that can contribute to the pathogenesis of AD (Xue et al., (2006) Neurosci Lett 405, 231-235).

  To investigate whether cellular stress alters BACE-1-AS nuclear retention, we used SH-SY5Y cells to depolarize KCl (30 mM for 5 min, a known hyperosmotic cell stressor). Exposed continuously). This cellular stress dramatically reduced the proportion of BACE-1-AS retained in the nucleus (FIG. 15B). This means that cellular stress releases BACE-1-AS into the cytoplasm. In order to exclude the possibility that this shift in BACE-1-AS nuclear retention after KCl treatment may be related to nonspecific poisoning and / or nuclear membrane disruption, we removed cells from KCl. And then subjected to a 60 minute recovery period in normal medium. The proportional pattern of antisense transcript concentration from nucleus to cytoplasm returned to baseline.

  There is significant evidence that Aβ1-42 has a strong cell stressor effect. Indeed, Aβ 1-42 enhances BACE-1 mRNA and protein activity, thereby damaging neurons through various cellular stress-related mechanisms (Tamagno et al., (2006) Free Radic Biol Med 41, 202-212). It has also been shown that direct injection of purified Aβ peptide into the murine brain results in protein oxidation and neuronal damage (Boyd-Kimball et al., 2005 Neuroscience 132, 313-324). Based on the above observations, we have observed that BACE- at a concentration (Tamagno et al., 2006) where Aβ1-42 is known to increase BACE-1 protein expression and thereby promote Aβ1-42 synthesis. It was hypothesized that 1-AS nuclear release could also be induced.

  Consistent with the above findings, synthetic Aβ1-42 peptide (1 μM for 2 hours) released BACE-1-AS from the nucleus, reversibly increasing its cytoplasmic concentration in SH-SY5Y cells (FIG. 15C). In addition, SH-SY5Y cells into conditioned medium from CHO-7PA2 cells (Walsh et al., 2005 Biochem Soc Trans 33, 1087-1090) that overexpress APP and contain significant levels of Aβ1-42 dimers and oligomers. 2 hours of exposure also elicited a similar translocation of the BACE-1-AS transcript from the nucleus to the cytoplasm. As a control, mock conditioned media from parental CHO cells did not induce any transition (FIG. 15D). Based on these findings, we propose that cellular stress and / or Aβ1-42 release BACE-1-AS from the nucleus. Given that the expression of BACE-1-AS and BACE-1 mRNA is consistently controlled, increased cytoplasmic BACE-1-AS increases the stability of BACE-1 mRNA and further Aβ1-42 production Is expected to result in a feedforward loop (FIG. 17). Such a mechanism may provide one explanation for Alzheimer's disease progression.

Stability of BACE-1 sense and BACE-1-AS antisense transcripts: Given the consistent relationship of BACE-1-AS and BACE-1 mRNA, BACE associated with cellular stress or exposure to Aβ1-42 An increased cytoplasmic concentration of -1-AS is thought to increase the stability of BACE-1 and thereby increase Aβ 1-42 production. To test this hypothesis and evaluate the potential mechanism by which increased cytoplasmic concentration of BACE-1-AS may enhance BACE-1 expression and function, we have determined that BACE-1-AS is stable for BACE-1 mRNA. Whether to increase the life and longevity was investigated. To measure the stability of the sense-antisense transcript, we blocked the synthesis of new RNA with α-amanitin (5 μg / ml), and four RNAs (18s rRNA, β-actin, BACE-1 and The change in the level of BACE-1-AS) was measured over 24 hours. BACE-1-AS has a shorter basal half-life than BACE-1 mRNA, and this finding is consistent with its regulatory properties (FIG. 16D). Furthermore, blocking RNA synthesis with α-amanitin confirmed that BACE-1-AS was the product of RNA polymerase II, further demonstrating our RACE data described above. Indeed, 18s ribosomal RNA, the product of RNA polymerase III, was not affected by α-amanitin treatment. Interestingly, during α-amanitin-mediated blockade of RNA polymerase II, Aβ1-42-mediated translocation of BACE-1-AS RNA significantly increases the stability of BACE-1 (FIG. 16E). Taken together, these findings suggest a model for regulation of BACE-1 mRNA by non-coding NAT through regulation of intracellular compartmentalization / release of NAT and the resulting sense transcript stability and longevity (FIG. 17).

  Increased levels of BACE-1-AS in the brain of APP transgenic mice: We have shown that increased levels of BACE-1-AS stabilize BACE-1 mRNA, thereby increasing BACE-1 expression and function It is postulated that this may contribute to increased Aβ 1-42 production and thereby promote the progression of pathophysiology associated with AD. If this is indeed the case, mice over-expressing APP with increased levels of Aβ1-42 should show increased cytoplasmic concentrations of BACE-1-AS. We next show that increased Aβ 1-42 levels in APP overexpressing mice (Li et al., (2004a) J. Neurochem 89, 1308-1312) alter the expression of BACE-1 sense or antisense transcripts. We investigated whether or not. Whole brain, cerebellum and liver were removed from 4 male APP mice and 4 corresponding wild-type mice 6 weeks old and used for protein and RNA extraction. As expected, the homogeneous time-resolved fluorescence (HTRF) assay showed that APP mice had significantly increased (˜300-fold) levels of Aβ 1-42 compared to wild type mice (FIG. 15E). . More importantly, in the brains of APP mice, BACE-1-AS transcripts were upregulated by about 45% and BACE-1 mRNA increased by about 25% compared to controls (FIG. 15F). . These findings are consistent with the following hypothesis: i) that BACE-1-AS and BACE-1 mRNA are consistently regulated, ii) elevated levels of Aβ1-42 are BACE- Associated with increased cytoplasmic concentration of 1-AS, and iii) increased levels of BACE-1-AS are associated with increased levels of BACE-1 and at the same time increased levels of Aβ1-42 That. Finally, it should be noted that the accumulation of Aβ1-42 in AD brain is a chronic process, and even a small positive feedback loop and an increase in BACE-1 expression can lead to a significant increase in amyloid deposition over time. (Li et al., (2006) Faseb J 20, 285-292).

Conclusion: We characterized a highly conserved nuclear-retained non-coding cis-antisense transcript for BACE-1, which consistently regulates the corresponding sense BACE-1 mRNA in vitro and in vivo Showed that. Multiple lines of evidence have been presented to support the idea that BACE-1 mRNA levels are consistently regulated by this natural antisense transcript. We also feed forward Alzheimer's disease progression in which cell stressors, including exposure to Aβ1-42, release this natural antisense transcript from the nucleus, thereby increasing BACE-1 activity and production of Aβ1-42. Provide evidence to support the model. This is, to our knowledge, the first report that directly linked non-coding RNA to the β-amyloid pathway.

Other Embodiments While the invention has been described in conjunction with the detailed description thereof, it is to be understood that the above description is intended to illustrate rather than limit the scope of the invention. Other aspects, advantages, and modifications are within the scope of the claims and their legal equivalents.
While the specification includes many specific embodiments, these are not limitations on the scope of the invention, but are to be construed as examples of preferred embodiments thereof. Many other variations are possible. All references cited herein are hereby incorporated by reference.

Claims (13)

A method for controlling the expression of a target gene comprising:
Targeting a nucleic acid molecule to a natural antisense transcript of the sense strand of the target gene, wherein the nucleic acid molecule targeting the antisense transcript is complementary to the antisense transcript; and Binding a nucleic acid molecule to the antisense transcript; wherein the antisense transcript is a non-coding RNA transcript and expression of the sense strand upon binding of the nucleic acid molecule to the antisense transcript And the expression of said gene is regulated;
Including
The target gene, Ru B ACE1 der,
Said method (however, excluding human treatment method).   2. The method of claim 1, wherein the nucleic acid molecule is an interfering RNA molecule. A composition for controlling expression of a target gene, comprising a nucleic acid molecule that targets a natural antisense transcript of a sense strand of a target gene, wherein the nucleic acid molecule that targets the antisense transcript complementary to the sense transcript, the antisense transcripts are non-coding RNA transcripts, wherein the nucleic acid molecule to control gene expression from the sense strand, wherein the target gene is Ru B ACE1 der, wherein Composition.   4. The composition of claim 3, wherein the nucleic acid molecule is an interfering RNA molecule.   The nucleic acid molecule is a ribozyme, small interfering RNA (siRNA), double stranded RNA (dsRNA), inverted repeat RNA, short hairpin RNA (shRNA), temporally controlled small RNA or clustering inhibitor RNA ( The composition according to claim 3, which is a ciRNA).   A modified or substituted at least one composed of linked regions that are chemically linked to one or more moieties or conjugates that are chimeric, wherein the nucleic acid molecule contains a modified backbone 6. The composition according to any one of claims 3 to 5, comprising one nucleobase and / or comprising a combination thereof.   7. The composition of any one of claims 3-6, wherein the nucleic acid molecule comprises a siRNA molecule that targets the overlap region of the sense / antisense locus. A composition according to any one of claims 3-7 for treating a neurological disease, the target gene is Ru der BACE1, the composition. Nerve disease including Alzheimer's disease, compositions of claim 8. Use of a nucleic acid molecule targeting a natural antisense transcript of a sense strand of a target gene for the treatment of a disease or condition, wherein the nucleic acid molecule targeting the antisense transcript is complementary to the antisense transcript a, said nucleic acid molecule to control gene expression from the sense strand, the antisense transcripts are non-coding RNA transcripts, wherein the target gene is Ru der BACE1, the use (although, the human Excluding treatment methods). 11. Use according to claim 10 , wherein the nucleic acid molecule is an interfering RNA molecule. Diseases or conditions including Alzheimer's disease Use according to claim 10. Nucleic acid molecules that regulate gene expression in one致的manner, to the use in medicine, to target and one nucleic acid molecule into the sense transcripts and antisense transcript of a gene even without small, the now A nucleic acid molecule targeting the sense transcript is complementary to the sense transcript, a nucleic acid molecule targeting the antisense transcript is complementary to the antisense transcript, and the antisense transcript is non- A coding RNA transcript, wherein the expression of the sense strand of the gene is inhibited and the expression of the gene is down-regulated upon binding of the at least one nucleic acid molecule to the antisense transcript and the sense transcript It said method comprising, the gene is B ACE1, the use.